Frequently Asked Questions

In general, InvivoGen’s cells are shipped on dry-ice and since dry-ice is not nearly as cold as liquid nitrogen, thawing of the cells technically begins during transport. Thus, we recommend a full thaw upon receipt to ensure the best recovery results.

NOTE: this is not applicable in some countries in Asia where cell lines are sent at room temperature using our specially designed flask

The most important step is the thawing procedure upon receipt of the cells. 

Do not put your cells at -80°C or in liquid nitrogen as this may damage the cells. 

You must propagate the cells immediately upon receipt.

Below are a few tips we recommend if you encounter any issues during initial culture:
• For the first 2-3 passages, grow cells in media containing 20% FBS and no antibiotics.
• Do not allow cells to reach 100% confluency. Please check the cells as regularly as possible.
• The cells should not be grown in 20% FBS for too long. Use media with 10% FBS after 2 or 3 passages.
• When making frozen stocks, continue growing additional cultures in case there is a problem with the frozen stock.

If you are working with THP1 cells in particular, here are a few additional tips:
• THP1 cells need to be passaged between 3 x 10⁵ and 7 x 10⁵ cells per mL (we recommend 5 x 10⁵ cells per ml). Below this, your cells will take a long time to grow and above 2 x 10⁶ cells per ml you may have toxicity.
• Between each passage, do not centrifuge the cells. THP1 cells grow better in conditioned media, therefore when passaging cells, leave 1 – 2 ml of the old media and add fresh media on top. Do not leave more than 50% of conditioned media as the cells will not have enough nutrients to properly grow.

After thawing, the cells can be more sensitive to selective antibiotics due to the initial low levels of resistance markers. Therefore, it is recommended to wait for 2 - 3 passages before adding the selective antibiotics in order to avoid further stress to the cells during the first couple of passages.

100 U/ml Penicillin and 100 µg/ml Streptomycin

No, this shouldn’t be a problem as long as there is at least 2 mM L-Glutamine in the medium. Most DMEM formulations already contain 4mM of L-Glutamine. However, if you need to add L-Glutamine to your medium you will only need to add 2 mM for the cells to grow properly.

Both Glutamine and GlutaMax have been tested in-house and can be used, with no difference in the cells noted.

Many of InvivoGen’s cell lines are engineered with a SEAP (Secreted Embryonic Alkaline Phosphatase) reporter system, and FBS, in many cases, contains traces of alkaline phosphatase (AP) that interferes with the test results. Therefore, to avoid background noise we recommend working with inactivated serum with all of our cells.

In house we inactivate serum by heating at 56°C for 30 minutes. It is important to wait for the water bath to reach the 56°C before placing the tube of serum in to ensure the serum is fully inactivated.

In house we always use heat-inactivated serum for the freezing medium, growth medium and test medium. However, please note that it is not a problem if you wish to use non-heat-inactivated in the freezing medium.

It is important to choose an endotoxin-free serum for the culture of our reporter cell lines. InvivoGen systematically asks for the Certificate of Analysis of different batches of serum to ensure a low level of endotoxin as to not activate the NFkB pathway.

When the HEK-Blue™ cells are non-adherent, either they were diluted too harshly at the start or they have grown over-confluent in a small flask and suffocated.
To avoid this in the future:
• Change the medium and seed the cells at a density of approximately 1.5 x 10⁶ cells/mL in a T25 flask.
• Wash the cells before putting them into a new flask. Sometimes when the cells are non-adherent, it is due to the clustering of both live and dead cells. Therefore, this will get rid of any remaining DMSO which could affect the adhesion of the cells to the flask.
• Use medium with 20% FBS.
• The use of CellBIND flasks can sometimes help to increase attachment and growth of the cells (however CellBIND flasks are not required in the normal protocol)

It isn’t normal for your cells to divide only once a week. Depending on the clone, they usually have a doubling time of 24 – 72 hours. THP1 cells must be cultured at quite a high concentration (at least 5 x 10⁵ cells/ml). Please note that our R&D teams have noticed that THP1 cells often die when they are diluted too harshly. Also, it may help to not add Normocin™ while waiting for improvement to their growth.

It is not a problem if the cells are clumping, as long as they are growing fine and have normal morphology. You may want to centrifuge the cells to get rid of the dead cells as sometimes this is the reason for clumping. Please note that we recommend homogenizing the cells before performing your assays.

We recommend to centrifuge at 120 G for 10 minutes.

It depends on the inhibitor. If the inhibitor blocks a receptor or an element in the signaling pathway, we recommend pre-incubating the inhibitor with the cells for at least 30 minutes. If the inhibitor is binding or blocking the ligand (i.e. an antibody against a specific cytokine) then it is best to pre-incubate the inhibitor with the ligand prior to adding to the cells.

In house the cells are seeded at the same time as adding the ligand (ligand first).

To limit the activation of NFκB before stimulation (lower background):
• Use healthy cells that have been passaged at least 24 hours before the assay
• Do not allow cells to be greater than 80 % confluent
• Use pre-warmed PBS to rinse your cells
• Use heat-inactivated FBS (to eliminate residual alkaline phosphatase in the serum)
• Do not centrifuge the cells prior to stimulation
• Rather than trypsin, use PBS for 2 - 3 min @ 37°C and gently pipet up and down to detach cells 
• Do not use an excessive number of cells per well
(approximately 50 000 cells/well as recommended on the TDS).

We recommend to not use Normocin™ in the test medium with all of our cell lines as it is better to reduce the number of potential interfering agents in the medium when performing the assay. The same goes for the use of selective antibiotics

It is hard to estimate the deviation factor regarding cell passage number. However, we note very little difference in our experimental results, with no more than the slight variations you expect due to handling errors.

Yes, InvivoGen’s reporter cell lines can be used in ELISA and Western Blots. However, please note that we do not sell them for this purpose.

They are semi-quantitative tests. However, they can be used for quantitative measurements by making a standard curve using a positive control.

If you cannot run the detection assays immediately, you can store supernatant samples for up to a week at 4°C. Supernatant samples are active for a longer time when kept at -20°C. However, in this case we would recommend supplementing the test medium with 20% FBS to protect the SEAP as much as possible during the freezing process. Do not repeat freeze/thaw cycles.

For adherent cells, we recommend DMEM, 20% (v/v) fetal bovine serum (FBS), and 10% (v/v) DMSO.

For suspension cells, we recommend fetal bovine serum (FBS) and 5-10% (v/v) DMSO

We highly recommend keeping a flask of cells running until you begin thawing your frozen stock in case something has gone wrong during the freezing step.

Our reporter cells require the activation of just one transcription factor (NFκB and/or AP-1) to produce a signal.

InvivoGen’s Lucia® luciferase is completely synthetic and is not related to any natural luciferase.

The difference in activity is approximately 10-fold.

Both cell types overexpress a designated TLR. The primary difference is that HEK-Blue™ cells include the NFκB inducible SEAP reporter construct.

HEK-Blue™ cells only express a single NFκB inducible SEAP reporter gene. Whereas, HEK-Dual™ cells have the addition of the Lucia™ gene knocked into the IL-8 locus. Thus, when IL-8 is activated following stimulation, HEK-Dual™ cells can report this with the secretion of Lucia™ luciferase.
It should also be noted that the HEK-Dual™ cells have been knocked out for TLR3, TLR5, and TNFR to limit interference from other TLRs when studying a specific TLR pathway.

HEK-Blue™ IL-1R cells express both human and murine IL-1β receptors, thus can detect both species.
On the other hand, HEK-Blue™ IL-1β cells are specific for human IL-1β, but can still detect higher concentrations of mouse IL-1β.

We presume them to be in the endosome as we express the wild-type, full-length genes. Additionally, their signal can be blocked by Chloroquine, an endosomal acidification inhibitor. However, as these TLRs are over-expressed there may potentially be low expression of them on the cell surface.

HEK293 cells express TLR1, TLR3, TLR5, TLR6, and NOD1.
They respond to TLR3, TLR5, and NOD1 agonists, but at a much lower level compared to HEK293 cells transfected with these receptors

In the United States, HEK293 cell lines are designated Biosafety Level 2 according to the Center for Disease Control and Prevention (CDC).
In Germany, HEK293 cell lines are designated Biosafety Level 1 according to the Central Committee of Biological Safety, Zentrale Kommission für die Biologische Sicherheit (ZKBS).
You can check with your country’s regulatory authority regarding the use of these cells.
Please note that there is no replicating/infectious Adenovirus 5 in these cells.

The minimal promoter isn't the same but the difference in expression for these two Null cell lines is minor.

There is no specific integration system used to generate our stable cell lines.
The selection pressure is enough to obtain stable clones. The receptors are added by simple transfection of plasmids using a cationic lipidic transfection agent (the plasmids are not linearized before transfection).

Only our HEK-Blue™ Null1-k and Null2-k cells are sensitive to G418.

The only HEK-Blue™ Null cells that are sensitive to Puromycin are the HEK-Blue™ Null1-v cells.

The only HEK-Blue™ Null cells that are sensitive to Puromycin are the HEK-Blue™ Null1-v cells.

Our RNAseq data confirms that our HEK-Blue™ cells express FcRN, however, this has not been functionally tested.

The split ratio will depend on when you expect confluency. Typically, the doubling time of HEK-Blue™ cells is approximately 24 hours.
Therefore, if you use a split ratio of 1:2 (50%) into a new flask, cells should be confluent the following day. If you use a split ratio of 1:4 (25%) you can expect the cells to be confluent after 2 days.

Our HEK-Blue™ Selection is provided in 1ml tubes with each containing a 250X solution.
Therefore, you should dilute HEK-Blue™ Selection 1:250 into your media to have a 1X concentration.

HEK-Blue™ cells should be seeded at a density of approximately 1.5 x 10⁶ cells in a T25 flask or 4 – 5 x 10⁶ cells in a T75 flask.

We recommend using a flat-bottom, clear walled cell culture plate.

Below are a few tips we recommend to help get your HEK cells growing:
• For the first 2-3 passages, grow cells in media containing 20% FBS and no antibiotics.
• Do not allow cells to reach 100% confluency Please check cells as regularly as possible.
• The cells should not be grown in 20% FBS for too long. Use media with 10% FBS after 2 or 3 passages.
• When making frozen stocks, continue growing additional cultures in case there is a problem with the frozen stock.

Trypsin does not adversely affect the health or growth of these cells. However, it is known that high concentrations will occasionally induce the activation of NFκB resulting in a higher background in your assay.
Moreover, we have observed some cases where trypsin has been contaminated with TLR2, TLR4, and TLR5 contaminants, which can also interfere with the assay results.

It is not unusual for different TLR cells to grow at different rates. Some TLR clones happen to grow a little slower/faster than others. This is often clone dependent.

When the HEK-Blue™ cells are non-adherent, either they were diluted too harshly at the start or they have grown over-confluent in a small flask and suffocated.
To avoid this in the future:
• Change the media and plate the cells at a density of approximately 1.5 x 10⁶ cells in a T25 flask.
• Wash the cells before putting them into a new flask. Sometimes when the cells are non-adherent, it is due to the clustering of both live and dead cells. Additionally, this will get rid of any remaining DMSO which could affect the adhesion of the cells to the flask.
• Use medium with 20% FBS.
• The use of CellBIND flasks can sometimes help to increase attachment and growth of the cells (however CellBIND flasks are not required in the normal protocol).

Yes, they can be used interchangeably. However, please note that the protocols are distinctly different and need to be followed accordingly.

HEK293 cells are very easy to transfect with a transfection efficiency of approximately 80%.

It depends on the cell line and the concentration of the ligand used to stimulate the cells. In general, we record the results following 16 – 24 hours of stimulation.

There are 2 possible explanations as to why a blue color is observed in all wells.

1. It could be due to the presence of Alkaline Phosphatase (AP) in the culture medium. To see if this is the case, there is a very simple test to perform. Add 50 µl of the medium used for cell culture (without cells) and 200 µl of resuspended HEK-Blue™ detection medium or QUANTI-Blue™. If the medium turns blue, then it is due to the presence of Alkaline Phosphatase (AP) in the serum of the media. In this case, you must heat the serum to inactivate the AP and repeat the medium test. At this point the test should give a negative result (no blue color).

2. It could be due to improper handling of cells before the test. To avoid activation of NFκB before stimulation and reduce the risk of false positive results:
• Use pre-warmed PBS to wash cells
• Use heat-inactivated FBS
• Do not centrifuge cells prior to stimulation
• Do not use trypsin

We have noticed a loss of sensitivity when using HEK-Blue™ Detection medium instead of QUANTI-Blue™ on our cytokine reporter cell lines.
Therefore, we recommend using QUANTI-Blue™, which is provided with the cells, as this is what we use in house.

We recommend to not use any antibiotics at all during assays to ensure the least amount of potential interfering agents in the medium.
Therefore, we do not add HEK-Blue™ Selection to the test media.

We have only tested the use of plasma and serum samples on our HEK-Blue™ hTLR2 cell line.
The results demonstrated that when compared to using standard samples (in DMEM), serum samples give a single log difference.
On the other hand, we found a 3-log difference between DMEM and plasma samples.
This is why we would recommend using serum samples over plasma samples.

THP1 cells display a high response to type I IFNs (alpha and beta), a lower response to type II IFNs (gamma) and no response to type III IFNs (lambda).

Below is a non-exhaustive list of THP1 ligand responses :
• TLR2: Good response through the NFκB pathway
• TLR3: Very low response through the NFκB pathway and a higher response (via TRIF) through the ISG pathway
• TLR4: Good response through the NFκB pathway
• TLR5: Good response through the NFκB pathway
• TLR7: No response
• TLR8: Good response through the NFκB pathway
• TLR9: No response

InvivoGen’s THP-1 cells also respond to NOD1, RIG-I, MDA-5 and STING/cGAS agonists.
(Please see the validation data sheet on the THP1-Dual™ cells webpage for more information)

These two cell lines have exactly the same sensitivity. The only difference is the reporting system.
Therefore, your choice will depend on if you prefer to monitor NFκB activation by determining the activity of SEAP, using a spectrophotometer, or Lucia™ luciferase using a luminometer.

THP1-Null cells were derived from the same parental clone of THP1 cells used to engineer all of InvivoGen’s THP1 reporter cells.
Additionally, they have been transfected with a mock plasmid, and are a proper control in terms of response and functionality.

The recommended concentration is between 3 – 7 x 10⁵ cells/ml depending on the cell line.
Generally, a concentration of 5 x 10⁵ cells/ml is used.

Below are a few tips we recommend to help get your THP1 cells growing:
• These cells need to be passaged between 3 x 10⁵ and 7 x 10⁵ cells/ml (we recommend 5 x 10⁵ cells/ml). Below this your cells will take a long time to grow and above 2 x 10⁶ cells/ml you may have toxicity.
• For the first 2 - 3 passages, grow cells in media containing 20% FBS and no antibiotics.
• Between each passage, do not centrifuge the cells. THP1 cells grow better in conditioned media, therefore when passaging cells, leave 1 – 2 ml of the old media and add fresh media on top. Do not leave more than 50% of conditioned media as the cells will not have enough nutrients to properly grow.
• The cells should not be grown in 20% FBS for too long. Use media with 10% FBS after 2 - 3 passages.
• When making frozen stocks, continue growing additional cultures in case there is a problem with the frozen stock.

It isn’t normal for your cells to divide only once a week. Depending on the clone, they usually have a doubling time of 24 – 72 hours.
THP1 cells must be cultured at quite a high concentration (~ 5 x 10⁵ cells/ml).
Please note that our R&D teams have noticed that THP1 cells often die when they are too dilute.
Also, it may help to not add Normocin™ while waiting for improvement to their growth.

It is not a problem if the cells are clumping, as long as they are growing fine and have normal morphology.
You may want to centrifuge the cells to get rid of the dead cells as sometimes this is the reason for clumping.
Please note that we recommend homogenizing the cells before performing your assays.

Treatment with phorbol myristate acetate (PMA, catalog #tlrl-pma) induces differentiation of THP1 cells into adherent macrophage-like cells.

Day 1
1- Add 180 µl of THP1 cell suspension per well of a 96-well plate (~ 100 000 cells/well).
2- Treat THP1 cells with 20 µl of PMA (final concentration 20 - 50 ng/ml) for 3 hours at 37°C in 5% CO2.
3- Wash cells gently with pre-warmed PBS and add 200 µl supplemented RPMI.
4- After 4-6 days (depending on the differentiation state of the cells) wash the cells with pre-warmed PBS and add 180 μl of supplemented RPMI.
5- Perform the reporter assay as per the TDS

We highly recommend waiting at least 72 hours before testing samples on the PMA-treated cells.
PMA is a specific activator of Protein Kinase C (PKC) and NFκB.
Therefore, it activates the production of SEAP which will result in a high background when using QUANTI-Blue™ for approximately 4 days following PMA treatment.

The production of pro-IL-1β can be further increased by priming PMA-activated THP1 cells with LPS.

THP1 cells should be at a density of approximately 7 x 10⁶ cells/ml before aliquoting into cryogenic vials.

For THP1 cells we recommend fetal bovine serum (FBS) and 10% DMSO.

Indeed, any selective antibiotics can be used together at the same time and in any combination.

We recommend not adding selective antibiotics until the cells have been passaged twice as cells are fragile during the initial culture procedure and the antibiotics may reduce cell viability. The resistance marker has to be expressed enough for the cells to acquire the resistance to these selective antibiotics.

For deactivation of all our selective antibiotics (Zeocin®, Hygromycin, G418, Phleomycin, Puromycin, and Blasticidin), we would recommend adding to the growth medium:

• - 5% NaOH (Sodium hydroxide)
• - 0.1% NaClO (Sodium hypochlorite)

You should then leave to react for 5 – 10 minutes and this should effectively deactivate all antibiotics in media.

Unfortunately we do not have data on the half-life of our selective antibiotics in cell culture medium. In house we prefer to add the selective antibiotics extemporaneously however if necessary you can supplement the medium with the antibiotic of your choice and keep at 4°C for no more than 1 week.

All of our selection antibiotics are shipped at room temperature. Upon receipt, they should be stored at 4°C or -20°C. Avoid repeated freeze-thaw cycles.
The expiry date is specified on the product label.
Once opened they remain stable for 1 year at -20°C providing you avoid repeated freeze-thaw cycles and they can be kept at 4°C for 3 months providing they are kept in sterile conditions.

We recommend to use Hygromycin B Gold at a concentration of 100 – 150 µg/ml for high salt LB conditions

Hygromycin B Gold is shipped at room temperature.
Upon receipt, it should be stored at 4°C or -20°C.
Avoid repeated freeze-thaw cycles.
The expiry date is specified on the product label. Once opened it remains stable for 1 year at -20°C providing you avoid repeated freeze-thaw cycles and it can be kept at 4°C for 3 months provided it is kept in sterile conditions.

The working concentrations of Hygromycin B Gold for mammalian cell lines vary from 50 to 200 μg/ml; in a few cases, up to 500 μg/ml. In a starting experiment we recommend to determine the optimal concentration of Hygromycin B Gold required to kill your host cell line. In general, Hygromycin B Gold is used at a concentration of 200 μg/ml, a 500-fold dilution from the stock solution. 
For more detailed information, please refer to the technical data sheet for this product.

Hygromycin-resistant transformants are selected in low-salt LB agar medium (yeast extract 5g/l, tryptone 10 g/l, NaCl 5 g/l, agar 15 g/l, pH 8) supplemented with 50 to 100 μg/ml of Hygromycin B Gold.
Plates containing Hygromycin B Gold are stable for 1 month when stored at 4 °C.

Zeocin-resistant transformants are selected in Low Salt LB agar medium (yeast extract 5 g/l, Tryptone 10 g/l, NaCl 5 g/l, Agar 15 g/l, pH 7.5) supplemented with 25 μg/ml of Zeocin®.
Note: Do not use an E. coli recipient strain that contains the Tn5 transposable element (i.e. MC1066). Tn5 encodes a bleomycin-resistance gene that will confer resistance to Zeocin®.

The Zeocin® we supply is hydrochloric.

No, Zeocin® is not photosensitive.

Zeocin® will undergo irreversible denaturation at high and low pH [6 - 8] or in the presence of a weak oxidant. 

Further dilutions to create an intermediate working solution are performed in sterile water.

The concentration of HEPES buffer used to reconstitute Zeocin® is 5 g/L .

Zeocin® is shipped at room temperature.
Upon receipt, it should be stored at 4°C or -20°C.
Avoid repeated freeze-thaw cycles.
The expiry date is specified on the product label.
Once Zeocin® solution opened, it remains stable for 1 year at -20°C providing you avoid repeated freeze-thaw cycles and it can be kept at 4°C for 3 months provided it is kept in sterile conditions.

The working concentration of Zeocin® for mammalian cell lines varies from 50 to 400 μg/ml, in a few cases can be as low as 20 μg/ml or as high as 1000 μg/ml. In a starting experiment, we recommend determining the optimal concentration of Zeocin® required to kill your host cell line.
For more detailed information, please refer to the technical data sheet for this product.

Phleomycin is shipped at room temperature.
Upon receipt, it should be stored at 4°C or -20°C.
Avoid repeated freeze-thaw cycles.
The expiry date is specified on the product label.
Once Phleomycin solution opened, it remains stable for 1 year at -20°C providing you avoid repeated freeze-thaw cycles and it can be kept at 4°C for 3 months provided it is kept in sterile conditions

The working concentration of phleomycin for mammalian cell lines varies from 5 to 50 μg/ml. In a starting experiment we recommend to determine the optimal concentration of phleomycin required to kill your host cell line. For more detailed information, please refer to the technical data sheet for this product.

The cells of the common E. coli recipient strains (i.e. HB101, DH5α, MC1061) transformed by vectors carrying bleomycin resistant genes, such as Sh ble and Tn5, are resistant to phleomycin.
Phleomycin-resistant transformants are selected in Low Salt LB agar medium (yeast extract 5g/l, Tryptone 10g/l, NaCl 5g/l, Agar 15 g/l, pH 7.5) supplemented with 5 μg/ml of phleomycin.

We actually observe the same phenomenon internally also. The explanation lies in the fact that even below 0°C, the freezing reaction of the solution does not start spontaneously: it needs a disturbance to start. This disturbance can be a vibration, a shock, etc...
Please note however that as soon as solidification has occurred in the liquid, it behaves like a chain reaction and propagates quickly throughout the available volume of solution.
According to our scientists, we often observe differences in solidification between several tubes of G418 which is explained by a small shock to a tube and not the others.

G418 is shipped at room temperature.
Upon receipt, it should be stored at 4°C or -20°C.
Avoid repeated freeze-thaw cycles.
The expiry date is specified on the product label.
Once opened it remains stable for 1 year at -20°C providing you avoid repeated freeze-thaw cycles and it can be kept at 4°C for 3 months provided it is kept in sterile conditions.

The working concentration of G418 Sulfate for selection and maintenance of mammalian cell lines transfected with the neo gene varies with a multitude of factors including cell type. In a starting experiment we recommend to determine optimal concentrations of antibiotic required to kill your host cell line by treating the cells with several concentrations, ranging from 100 μg/ml to 1 mg/ml.
Please note that G418 is normally used at a concentration of 400 μg/ml.

No. G418 is used to select mammalian cells expressing the Neo gene. 
The appropriate antibiotic for selection in E. coli expressing the Neo gene is Kanamycin.

During storage a crystalline precipitate of puromycin may form. If this occurs, heat the product at 37 °C for 30 minutes until the crystalline precipitate disappears. The formation of a crystalline precipitate does not affect the activity of the product.

Puromycin is shipped at room temperature.
Upon receipt, it should be stored at 4°C or -20°C.
Avoid repeated freeze-thaw cycles.
The expiry date is specified on the product label.
Once opened it remains stable for 1 year at -20°C providing you avoid repeated freeze-thaw cycles and it can be kept at 4°C for 3 months provided it is kept in sterile conditions.

The working concentrations of puromycin for mammalian cell lines range from 1 to 10 μg/ml. In a starting experiment we recommend to determine optimal concentrations of antibiotic required to kill your host cell line. Puromycin quickly kills eukaryotic cells that do not contain the pac gene. For more detailed information, please refer to the technical data sheet for this product.

Puromycin is poorly active on E.coli. However, puromycin-resistant transformants can be selected in LB agar medium supplemented with 100-125 μg/ml of puromycin [pH adjusted to pH 7.5 - 8].

Blasticidin is shipped at room temperature.
Upon receipt, it should be stored at 4°C or -20°C.
Avoid repeated freeze-thaw cycles.
The expiry date is specified on the product label.
Once Blasticidin solution opened, it remains stable for 1 year at -20°C providing you avoid repeated freeze-thaw cycles and it can be kept at 4°C for 3 months provided it is kept in sterile conditions.
Regarding Blasticidin powder, the stock solution must be at a concentration of 10 mg/ml and aliquots can be kept 1 year at -20°C (below 10 mg/ml we cannot guarantee the stability of the product). Again, avoid repeated freeze-thaw cycles.

The working concentration of Blasticidin for mammalian cell lines varies from 1 to 10 μg/ml, in a few cases up to 30 μg/ml. In a starting experiment we recommend to determine optimal concentrations of antibiotic required to kill your host cell line. For more detailed information, please refer to the technical data sheet for this product.

E.coli is poorly sensitive to blasticidin, but transformants resistant to blasticidin can be selected on low salt LB agar medium (pH 8) supplemented with 100 μg/ml blasticidin. High pH enhances the activity of blasticidin

Firstly, ensure that you are working in a sterile environment and using proper aseptic techniques. It is also important to avoid talking over your cells and sneezing. Secondly, quarantine any incoming cell cultures until these have been confirmed free of contamination. Thirdly, monitor your cell cultures for contamination on a regular basis by optical microscopy and detection kits. For example, InvivoGen provides MycoStrip™ and PlasmoTest™ for the detection of mycoplasma contamination in cell cultures. Lastly, you can use antibiotic cocktails, such as those offered by InvivoGen, specifically designed for taking a preventive strike against microbes that would be difficult to detect in new cultures (i.e. primary cells, or cloning), such as Plasmocin® prophylactic, Normocin™, Primocin® or Fungin™.

Bacteria and fungi can usually be identified by optical microscopy. Their fast growth rate allows their detection by the naked eye as early as 48 hours (i.e. over the weekend), the contaminated cultures appearing turbid or spotty. Subsequently, identification of these microorganisms can be performed with testing kits. Mycoplasma in cell cultures cannot be detected visually, not even by optical microscopy. Hence, these microbes can go unnoticed for long periods and can only be identified using dedicated assays. Detection techniques (such as MycoStrip® and PlasmoTest™) can be used in tandem to ensure accurate analysis, especially to rule out false positives or clarify ambiguous results.

All depends on the cell line you have in culture and if you are looking to prevent infection from a specific type of contaminant or a broader range of contaminants.
For example, if you are specifically looking to prevent fungal contaminations from your cell cultures, we would recommend using our Fungin™. If you are looking to prevent mycoplasma contamination, we have Plasmocin® prophylactic.
If on the other hand you are looking for to prevent infection from a broad range of contaminants, we would recommend Normocin™ which is designed to prevent cell lines from mycoplasma, bacterial and fungal contaminations. We also have Primocin® which is specifically designed to offer complete protection of primary cells from microbial contamination.

Yes, it is even recommended to use either Plasmocin® prophylactic or Normocin™. 
Please note however that it is necessary to check that the cells are not contaminated beforehand.

We actually provide two antibiotics for this purpose, Normocin™ and Primocin®. Both antibiotics provide complete protection from microbial contamination, however, Primocin® was specifically designed for the protection of primary cells whereas Normocin™ is recommended for cell lines.

No, they do not interfere with common selective antibiotics such as G418, Blasticidin, Puromycin, Hygromycin B, and Zeocin®.

Yes, InvivoGen provides a free guide dedicated to this topic: Read our Pratical guide (.pdf).

In the early stages of contamination, bacteria can be mistaken for cellular debris as they are much smaller (1-10 μm) than eukaryotic cells (10-100 μm). Therefore, it is important to check your cell cultures under a light microscope using phase contrast (100x - 400x). If you observe the abnormal presence of small black dots, rods, spirals, either alone, in chains, or clusters, your culture is probably contaminated. Because of their fast growth rate, bacteria cause a change in the culture medium in just 48 hours, making the contamination clearly visible with the naked eye from 10^5 cfu/ml. The culture medium appears cloudy, and if it contains phenol red, a rapid colour change from red to yellow indicates a decrease in pH, a consequence of bacteria metabolism. The culture environment is no longer suitable for eukaryotic cells leading eventually to their death. These cultures should be discarded, or if irreplaceable, treated with InvivoGen’s antibiotic cocktails such as Plasmocin® Treatment, Normocure™ or Plasmocure™.

InvivoGen provides a range of anti-bacterial agents to prevent contamination of your precious cell lines. First we have Normocin™ which is an innovative formulation of three antibiotics active against mycoplasma, bacteria (Gram+ and Gram-), and fungi, including yeasts. It is widely used and cited as a “routine addition” to cell culture media to prevent bacterial contamination.
Next we have Primocin® which contains four compounds, with three of these blocking DNA and protein synthesis in Gram+ bacteria, Gram- bacteria, and mycoplasma. Primocin® was specifically designed for the protection of primary cells whereas Normocin™ is recommended for cell lines.
Finally, we also have Plasmocin® prophylactic which contains two potent bactericidal components: one that acts on protein synthesis machinery, and one that acts on DNA replication. Although originally designed to target mycoplasmas, it is also active against other types of bacteria.

Normocure™ is the best weapon to save your valuable cell lines from Gram+ and Gram- bacteria, especially non-fermenting Gram- bacteria that are resistant to Pen-Strep and Normocin™. Normocure™ is a cocktail of three components belonging to different antibiotic families. After the first passage, > 99% of bacterial contaminants are eliminated. The targets of these antibiotics are absent in eukaryotic cells, ensuring Normocure™’s low cytotoxicity.
Please note that we also have Plasmocin® Treatment and Plasmocure™ which can be used for bacterial contaminations. Although these products are designed to target mycoplasmas, they are also active against certain types of bacteria.

We would highly recommend using Normocure™ which is a broad-spectrum antibacterial agent highly effective against Gram+ and Gram- bacteria. Cell cultures contaminated with bacteria from the environment, such as Staphylococcus species and Achromobacter species, can be efficiently cured by Normocure™ treatment.

Hyphae can be detected by the naked eye or a light microscope depending on their size and growth stage. Yeasts are the smallest form (3-10 μm) of fungi. They can be seen using a light microscope.
In the case of substantial contamination, colonies form as molds floating on the surface. In this case, do not open the vessel and discard the cultures to avoid spreading spores. Sometimes, the medium pH may increase, resulting in phenol-red containing media to appear pink.

For both the protection and elimination of fungal contaminations in your cell cultures, InvivoGen provides Fungin™ which is a highly referenced antimycotic reagent. Fungin™ kills the different forms of fungi (yeasts, hyphae, and molds) by disrupting ionic exchange through the cell membrane.

Fungin™ may be added to media containing commonly used antibacterial agents, such as Pen-Strep.

You should have no problem using our antifungal reagent, Fungin™ in other media such as blood agar and MacConkey agar.

We would recommend continuing treatment on one half of the culture for at least another 3 – 4 days. On the other half of the culture, change the medium with fresh medium that no longer contains any antibiotics (no Fungin™ or Pen-Strep) and spread 1 mL of the culture medium on Sabouraud agar to determine if the fungal contamination is still present. For this you should leave the petri dish at 20 - 25°C and check after 3 – 5 days if fungi appears.

On the technical data sheet we state that Fungin™ should be used at 50 µg/ml but we would recommend using it in parallel at 75µg/ml (seeing as it is not very toxic for cells) to estimate the best concentration for fungi removal. The treatment should last 5 – 10 days.

Mycoplasma in cell cultures cannot be detected visually, not even by optical microscopy. Hence, these microbes can go unnoticed for long periods and can only be identified using dedicated assays, ie MycoStrip™ and PlasmoTest™

Plasmocin® prophylactic was specially designed for the prevention of mycoplasma contaminations. It contains two potent bactericidal components: one that acts on protein synthesis machinery, and one that acts on DNA replication.
We also provide Normocin™ and Primocin® which provide complete protection from microbial contamination (such as mycoplasma, bacteria and fungi).
Please note however that Primocin® was specifically designed for the protection of primary cells whereas Normocin™ is recommended for cell lines.

InvivoGen offers two different kits for the detection of mycoplasma, PlasmoTest™ and MycoStrip™. PlasmoTest™ and MycoStrip™ allow rapid and accurate detection of cell culture contamination by mycoplasma as ‘routine’ and ‘occasional’ procedures, respectively.
PlasmoTest™ is a cell-based colorimetric assay which, in the presence of mycoplasma contaminated samples, induces the secretion of Alkaline Phosphatase which is easily detected in culture medium. The hands-on time is about 30 minutes and the results are obtained after an overnight incubation. This kit is specially designed for labs doing a lot of cell culture and can be established as a routine procedure in the lab. The advantage of this kit is that it is highly cost-effective as the critical component of the PlasmoTest™ system is an engineered cell line that can be propagated to make frozen stocks for unlimited use. Once you have the PlasmoTest kit you only need to purchase the HEK-Blue Detection media, reducing drastically the cost per sample.
Additionally, InvivoGen has designed a new and innovating mycoplasma detection assay, MycoStrip™, based on isothermal PCR. Results are visualized as a band on an lateral flow detection strip within 5 minutes. This kit is specially designed to provide the user with minimal hands on time and rapid and clear results – with no need for calculation, distinguishing similar colors, or running of an agarose gel. Additionally, the only equipment needed to perform the assay is a heat block.

Detection of mycoplasma by InvivoGen’s MycoStrip™ is based on isothermal PCR, a robust technique that exponentially amplifies DNA at a constant temperature of 65°C. Using our proprietary Reaction Mix, the 16S rRNA gene in the most commonly found mycoplasma species in cell culture, accounting for 95% of contamination, is targeted and amplified. Results are rapidly visualized as a band on an lateral flow detection strip.

PlasmoTest™ is a cell-based colorimetric assay that exploits the ability of Toll-like receptor 2 to recognize mycoplasmas and to induce a signalling cascade leading to the activation of NF-κB and other transcription factors. In the presence of mycoplasmas, TLR2 expressed on the surface of HEK-Blue™-2 cells activates these transcription factors which in turn induce the secretion of sAP (secreted alkaline phosphatase), a reporter protein easily detectable by the purple/blue coloration of the HEK-Blue™ Detection medium.

Mycoplasma contamination can be efficiently and rapidly eliminated with the Mycoplasma removal agents, Plasmocin® or Plasmocure™. They combine two antibiotics that act through different mechanisms and allow mycoplasma eradication in only 2 weeks.

Plasmocin® and Plasmocure™ are cocktails of different antibiotics, both of which will allow you to get rid of your mycoplasmas completely. Please note however that we recommend starting treatment with Plasmocin®.
(We recommend using Plasmocure™ only in case of resistance to Plasmocin®, which is extremely rare).

If both Plasmocin® and Plasmocure™ did not work then it seems like your sample is too contaminated.
The best option, in this case, is to dilute the cells as much as possible at the next passage (this way you will also dilute the quantity of contaminants).
Then we recommend using Plasmocure™ at different concentrations (to be sure that it is not too toxic for the cells) for 2 weeks. After the 2 week treatment, split the cells in two and on one half continue the treatment for another week and on the other half, wait three days without using any antibiotics and then check for the presence of the contaminant, using for example PlasmoTest™. If you are still contaminated, please contact us.

Plasmocin® is active on both extracellular and intracellular mycoplasma.

Treatment in a T25 flask is suitable.
Please note that the treatment is most effective when the cells are “passaged” as much as possible (to remove mycoplasma and add fresh Plasmocin® or Plasmocure™) and also when the cells are not too confluent (60 - 70% max).

It seems like all mycoplasmas were not killed following the treatment. To prevent this from happening again, we recommend dividing your culture in half after the two weeks of treatment with Plasmocin®.
On one half you continue the treatment for another week (without interruption) to prevent the mycoplasmas from developing again if they are still present in a basal manner. On the other half, we recommend to stop the treatment and grow your cells 3 days without Plasmocin® or any antibiotics at all.
After the 3 days of culture, you can then perform your mycoplasma detection test which should detect any residual mycoplasma that was left after the two week treatment.

Plasmocin® is suitable for Sf9 cells, and should suit other insect cells (but unfortunately we do not have additional data on other insect cells).

The LPS detection kit is highly sensitive and can detect as little as 0.01 EU/ml.

The use of some FBS might affect the functionality of HEK-Blue™-4 Cells as they may contain endotoxins which would interfere with your assay. Make sure the FBS used is endotoxin-free. To avoid having endotoxin contaminants, we recommend to ask for manufacturer certification of endotoxin levels before purchasing the FBS to ensure it is endotoxin-free.

The cell line should be propagated upon receipt to make your frozen stocks for further use (no expiry date as the cells can be kept a very long time in liquid nitrogen). Regarding the rest of the kit, it should remain stable for 1 year (providing that it is stored in the correct conditions)

There are 2 ways to avoid this nonspecific activation:
- You can either use our parental cell line as a negative control, the HEK-Blue™ Null1 cells (this cell line only expresses the NFkB inducible system). Therefore, any non-specific activation of NFkB will also be detected with the HEK-Blue™ Null1 cells.
- Otherwise, you can antagonize the TLR4 response with the Ultrapure LPS-RS which is an antagonist of LPS. If with this antagonist you eliminate the signal, this will then confirm that it is a specific TLR4 activation of your sample.
The reference of this antagonist is the following: LPS-RS ultrapure (#tlrl-prslps).

Endotoxins, also known as lipopolysaccharides (LPS) or lipoglycans, are a major cell wall component of Gram-negative bacteria. Endotoxins are potent inducers of inflammatory responses both in vitro and in vivo. Endotoxin contaminations are a major concern for cell cultures and production of injectable drugs. Thus, extra-care needs to be taken with solutions and reagents that are sterile but may still contain bacterial components, and monitoring for the presence of endotoxins in cell culture reagents is crucial.

Sources of endotoxins include media, sera, water, buffers and other cell culture reagents, such as trypsin. To avoid having endotoxin contaminants, we recommend to ask for manufacturer certification of endotoxin levels, or test before use using our HEK-Blue™ LPS Detection kit 2.

The HEK-Blue™ LPS Detection Kit is a cell-based colorimetric assay for the detection of biologically active endotoxin. This kit is based on the ability of TLR4 to recognize structurally different LPS from gram-negative bacteria.
The presence of minute quantities of LPS, starting as low as 0.01 EU/ml, are detected by the HEK-Blue™-4 cells leading to the activation of NF-κB. Using HEK-Blue™ Detection, a specific detection medium, NF-κB activation can be observed with the naked eye or quantified by reading the OD at 650 nm.
You can find further details on the following document: HEK-Blue™ LPS Detection Kit (.pdf).

With the LPS detection kit 2, we provide 1 ml of QB reagent and 1 ml of QB buffer (sufficient to prepare 100 ml of QUANTI-Blue™ Solution, a SEAP detection reagent).
This should be sufficient to perform assays in approximately five 96-well plates (480 wells).

No, Primocin® does not contain Tetracycline.

Primocin® is a preventive antibiotic, active against both Gram+ and Gram- bacteria, mycoplasma and fungi. In other words, Primocin® will NOT induce the elimination of microbial contamination in already infected cells.

Primocin® is shipped at room temperature.
Upon receipt it can be stored at 4°C for 3 month or at -20°C for long-term storage.
Avoid repeated freeze-thaw cycles.
The expiry date is specified on the product label.
Once opened it remains stable for 1 year at -20°C providing you avoid repeated freeze-thaw cycles.

Both antibiotics provide complete protection from microbial contamination however Primocin® was specifically designed for the protection of primary cells whereas Normocin™ is recommended for cell lines.

Yes, Normocin® is one of the antibiotics we use as a standard antimicrobial agent.
Our cell lines have been developed with this antimicrobial agent so we prefer to use it in house.

Yes, in house we use Normocin® for hybridoma culture.

Normocin® is shipped at room temperature.
Upon receipt, it should be stored at 4°C or -20°C.
Avoid repeated freeze-thaw cycles.
The expiry date is specified on the product label.
Once opened it remains stable for 1 year at -20°C providing you avoid repeated freeze-thaw cycles and it can be kept at 4°C for 3 months provided it is kept in sterile conditions.

Normocin™ is a formulation of three antibiotics active against mycoplasmas, bacteria and fungi. Plasmocin® prophylactic on the other hand is a cocktail of 2 different antibiotics, it is a specific anti-mycoplasma reagent.

Both antibiotics provide complete protection from microbial contamination however Primocin® was specifically designed for the protection of primary cells whereas Normocin® is recommended for cell lines.

Normocin® does contain Phenol Red. If you cannot use an additive with Phenol Red and would like a broad-spectrum antibiotic/antimycotic/anti-mycoplasma agent, you can use our Primocin®.

Normocure™ is shipped at room temperature.
Upon receipt it can be stored at 4°C for 3 month or at -20°C for long-term storage.
Avoid repeated freeze-thaw cycles.
The expiry date is specified on the product label.
Once opened it remains stable for 1 year at -20°C providing you avoid repeated freeze-thaw cycles.

Yes, Normocure™ is a broad-spectrum antibacterial agent highly effective against Gram+ and Gram- bacteria. Cell cultures contaminated with bacteria from the environment, such as Staphylococcus species and Achromobacter species, can be efficiently cured by Normocure™ treatment.

The cloudy precipitate is one of the two antibiotics in Plasmocin® coming out of solution after freeze-thawing. 
The antibiotic is basically at the limit of its solubility at the concentration provided and thus freeze-thawing causes it to come out of solution. 
We don’t recommend sonicating. Letting it sit at 37 °C for 30 minutes - 1 hour and then vortexing for a minute or two should get it back into solution. 
Plasmocin® is stable for 1 week at 37 °C so there is no concern about heating it for an hour to get the precipitate back in solution. Alternatively you can make a more dilute stock (12.5 mg/ml) by making a 1:1 dilution with sterile water thus increasing the volume in which the antibiotic is dissolved in.

We do not recommend using Plasmocin® with other antibiotics (such as gentamycin and fungizone™) as this may interfere. Plasmocin® can be toxic in some cells therefore we prefer not using additional antibiotics with this treatment that could end up overwhelming sensitive cell lines.

Plasmocin® Treatment is shipped at room temperature.
Upon receipt it can be stored at 4°C for 1 month or at -20°C for long-term storage.
Avoid repeated freeze-thaw cycles.
The expiry date is specified on the product label.
Once opened it remains stable for 1 year at -20°C providing you avoid repeated freeze-thaw cycles.

If possible, we highly recommend starting treatment on a fresh batch of cells that haven’t been treated with another anti-mycoplasma agent to be sure that your cells aren’t resistant to one of the antibiotics present in Plasmocin® Treatment.

Plasmocure™ is shipped at room temperature.
Upon receipt it can be stored at 4°C for 12 months or at -20°C for long-term storage.
Avoid repeated freeze-thaw cycles.
The expiry date is specified on the product label.
Once opened it remains stable for 1 year at -20°C providing you avoid repeated freeze-thaw cycles and it can be kept at 4°C for 3 months provided it is kept in sterile conditions and at a concentration of 100 mg/ml.

Plasmocin® and Plasmocure™ are cocktails of different antibiotics, both of which will allow you to get rid of your mycoplasmas completely. Please note however that we recommend starting treatment with Plasmocin®. (We recommend using Plasmocure™ only in case of resistance to Plasmocin®, which is extremely rare).

If both Plasmocin® and Plasmocure™ did not work then it seems like your sample is too contaminated.
The best option, in this case, is to dilute the cells as much as possible at the next passage (this way you will also dilute the quantity of contaminants).
Then we recommend using Plasmocure™ at different concentrations (to be sure that it is not too toxic for the cells) for 2 weeks. After the 2 week treatment, split the cells in two and on one half continue the treatment for another week and on the other half, wait three days without using any antibiotics and then check for the presence of the contaminant, using for example MyocStrip™ or PlasmoTest™.
If you are still contaminated, please contact us.

There is no CAS number for Plasmocin® Prophylactic seeing as it is a cocktail of different antibiotics

There are no calcium salts in Plasmocin® prophylactic.

No, Plasmocin® does not contain Tetracycline.

Plasmocin® prophylactic is shipped at room temperature.
Upon receipt it can be stored at 4°C for 1 month or at -20°C for long-term storage.
Avoid repeated freeze-thaw cycles.
The expiry date is specified on the product label.
Once opened it remains stable for 1 year at -20°C providing you avoid repeated freeze-thaw cycles.

Fungin™ is shipped at room temperature.
Upon receipt, it should be stored at 4°C or -20°C.
Avoid repeated freeze-thaw cycles.
The expiry date is specified on the product label.
Once opened it remains stable for 1 year at -20°C providing you avoid repeated freeze-thaw cycles and it can be kept at 4°C for 3 months provided it is kept in sterile conditions and at a concentration of 10 mg/ml.

No, Fungin™ does not contain Amphotericin B. Unlike Amphotericin B, Fungin™ is a highly stable compound and it does not need to be dissolved in toxic deoxycholate.

Fungin™ may be added to media containing commonly used antibacterial agents, such as Pen-Strep.

You should have no problem using our antifungal reagent, Fungin™ in other media such as blood agar and MacConkey agar.

We would recommend continuing treatment on one half of the culture for at least another 3 – 4 days. 
On the other half of the culture, change the medium with fresh medium that no longer contains any antibiotics (no Fungin™ or Pen-Strep) and spread 1 mL of the culture medium on Sabouraud agar to determine if the fungal contamination is still present. For this you should leave the petri dish at 20 - 25°C and check after 3 – 5 days if fungi appears.

On the technical data sheet we state that Fungin™ should be used at 50 µg/ml but we would recommend using it in parallel at 75µg/ml (seeing as it is not very toxic for cells) to estimate the best concentration for fungi removal. The treatment should last 5 – 10 days.

Yes, it is possible to use water instead of PBS.

Detection of mycoplasma by InvivoGen’s MycoStrip® is based on isothermal PCR, a robust technique that exponentially amplifies DNA at a constant temperature of 65°C. Using our proprietary Reaction Mix, the 16S rRNA gene in the most commonly found mycoplasma species in cell culture, accounting for 95% of contamination, is targeted and amplified. Results are rapidly visualized as a band on an immunochromatographic strip.

If you do not have a thermocycler, you can use a calibrated heatblock or a water bath instead. Please note that the use of a water bath can introduce new sources of contamination.

It is important to include positive and negative controls on a regular basis to monitor the reliability of your results and in case of troubleshooting.

Exceeding the 40-minute incubation at 65°C significantly impacts the results of the assay and therefore, we do not recommend to use this sample.

The positive control is not a source of mycoplasma contamination; it only contains a fragment of DNA.

As isothermal PCR is a very sensitive method, heatblock must be precisely at 65°C.
A higher temperature will inhibit the reaction.
We recommend to control heatblock temperature or switch to a thermocycler.

There is no need to worry, the presence of mammalian cell DNA will not affect the detection assay

Samples can be stored as followed:
Cell culture supernatant: before starting the “preparation of samples” protocol, you can store the cell culture supernatant at -20°C until required.
Before use, completely thaw the sample at 37°C and mix well by vortexing to ensure all salts are dissolved.

Prepared sample: After performing steps 1 – 4 of the “preparation of samples” protocol, your sample can be stored at -20°C until required.

Processed sample: After performing steps 1 – 4 of the “detection assay” protocol, the processed samples can be stored at -20°C for up to 3 months.

If you observe a faint band for one of your samples, we consider this to be a positive result for mycoplasma contamination. We recommend to confirm this result using one of the following tips:
• Concentrate your sample: begin with a larger volume of supernatant and/or repeat steps 2-3 in the “preparation of samples” protocol. Re test the concentrated sample using MycoStrip®.
• Continue to grow the culture for an additional 48 hours before re-testing using MycoStrip®.
• Use sterile water to resuspend the sample instead of PBS (to increase the sensitivity of the test).

MycoStrip® is highly sensitive having been validated to detect as low as 10² genome copies per ml.

MycoStrip® does not detect any of the phylogenetically related microorganisms, such as Clostridium, Lactobacillus, and Streptococcus. Likewise, the waterborne germ Burgholderia is not detected. The specificity of MycoStrip® has been assessed on DNA extracts of Mollicutes, non-Mollicute bacteria, and eukaryotic cell/tissue samples. Additionally, the cross-reactivity with eukaryotic DNA was not observed. Read the complete list of all species detected and tested.

Contrary to other mycoplasma detection kits, only a thermocycler is needed!

The sample preparation requires only 10 minutes and the detection assay takes 60 minutes including 10 minutes hands on time. Results are visualized as a band within 5 minutes.

The MycoStrip® test requires 1 mL of cell culture supernatant from suspension or adherent cell cultures prior to passaging, or cell culture media constituents such as fetal bovine serum (FBS).

MycoStrip® is a robust, sensitive, and extremely simple method for the detection of mycoplasma. The speed and convenience of MycoStrip® allows for routine testing of cells in culture and commonly used constituents of complete media (e.g. FBS). It requires minimal hands-on-time with rapid results which are easily interpreted – no need for calculation, distinguishing similar colours, or running of an agarose gel.

The kit is shipped at room temperature.
Upon receipt, store all components at -20°C.
All components, including the strips, remain stable for 12 months when properly stored.

No, the use of antibiotics on your cells will not interfere with the assay.

There is no increase to sensitivity however this step will kill the mycoplasma avoiding the handling of live mycoplasma.

Actually, the positive control is a translucid lipidic film that is often difficult to observe with the naked eye. The negative control on the other hand is a visible powder.

We have little experience testing plasma and serum samples however it has been performed on our HEK-Blue™-2 cells in the PlasmoTest™ kit. The results show that when compared to using standard samples (in DMEM), serum samples give a single log difference (example: in serum we detect up to 10^4 UFC/ml of Mycoplasmae Fermentans whereas in DMEM we detect up to 10^3 UFC/ml of M. Fermentans).
On the other hand, we found a 3-log difference between DMEM and plasma samples (example: in plasma we detect up to 10^6 UFC/ml of M. hominis whereas in DMEM we detect up to 10^3 UFC/ml of M. hominis). This is why we would recommend using serum samples over plasma samples.

The most common mycoplasma detection test used is PCR, but this technique has its drawbacks, notably if you do not perform PCR regularly in your lab this might be an issue due to the stability of the taq polymerase, sterility of all other PCR reagents such as dNTPs, buffer...
PlasmoTest™ is the first cellular assay for the visual, colorimetric detection of mycoplasma contamination in cell cultures. It requires only basic cell culture knowledge and a hands-on time of under 1 hour with results after overnight incubation. Most importantly, a positive result indicates the presence of a cell culture contaminant so there are no false positives.

PlasmoTest™ detects the presence of mycoplasma lipoproteins, thus it indicates the presence of both live and dead mycoplasma in the cell culture.

PlasmoTest™ relies on the activation of TLR2. Therefore, it can detect both mycoplasma and bacteria contaminants. However, while the mycoplasma cannot be detected by the naked eye, bacteria contamination is visible and leads to a decreased pH (change of medium colour to yellow) and medium turbidity due to bacterial growth.

Any wavelength between 620 nm and 655 nm is optimal to read the absorbance. Please note that for readers that cannot get to this part of the spectrum, you can get a signal from 600 – 680 nm. Based on our analysis of QUANTI-Blue with and without SEAP, looking at absorbance from 350 – 700 nm we find that 600 nm up to 680 nm will yield a signal, however there will be a lower saturation and potentially a higher background

It is important to collect a few cells with the supernatant as this will enable the detection of intracellular mycoplasma, if there are any as well as the extracellular mycoplasma which is in the supernatant.

All samples must be heated in order to get rid of any potential Alkaline Phosphatase present in the serum of your supernatants to be tested which can interfere with your results. The heating step also allows the elimination any remaining mycoplasma in this sample to avoid spreading of the contamination. You can however try testing just the test medium used for cell culture (without cells) and resuspended HEK-Blue™ detection. If the medium does not turn blue then you can skip the heating step but please be very careful with this potentially contaminated sample.

We cannot disclose any information on these controls but please be assured that the positive control is not live mycoplasma.

When developing PlasmoTest™ we have shown that some Trypsin or serum are able to induce a TLR2 response (from sterile contaminations) but never from PBS. DMSO over 1/1000 will be toxic for the HEK Blue cells which can induce relatively high background noise. It is useful to test the medium alone in parallel with your supernatant as a negative control.

There are 3 possible explanations as to why you observe a blue colour in all wells: 
1. It could be due to the presence of Alkaline Phosphatase (AP) in the culture medium. To see if this is the case, there is a very simple test that can be performed. Just add 50 µL of the medium used for cell culture (without cells) and 200µL of the resuspended HEK-BlueTM detection. If the medium turns blue then it is due to the AP in the serum of the media. In this case, you must heat up the culture medium to eliminate the AP and redo the same test. At this point the test must not be blue (medium + detection medium should give a negative result).

2. If the medium does not react with the HEK-BlueTM detection medium, then the problem might be due to the cells (HEK-BlueTM -2 cells) which are contaminated. In this case, the best option would be to start from fresh with a new vial of cells (if you have remaining stocks). Also, please note that it is best to use a pipet with filter-tips to avoid any contamination of the cell suspension.

3. It could be due to improper handling of cells before the test. Here are a few tips in order to lower the background level (to limit the activation of NFkB before stimulation) and therefore limit the risks of false positive results:
- use pre-warmed PBS to wash cells
- use heat inactivated FBS (some lots of FBS, although sterile, contain microbial debris that may activate the HEK-Blue™-2 cells)
- do not centrifuge cells prior to stimulation
- do not use trypsin

• DO (positive control): 0.9 – 1.2
• DO (negative control): < 0.2

If the sample was sterile and kept sterile at room temperature your results will be the same.
However it is recommended to freeze your samples at -20°C (for several days) if they can’t be tested right away.

The split ratio will depend on when you expect confluency. Typically, the doubling time of HEK-Blue™ cells is approximately 24 hours.
Therefore, if you use a split ratio of 1:2 (50%) into a new flask, cells should be confluent the following day. If you use a split ratio of 1:4 (25%) you can expect the cells to be confluent after 2 days.

With the LPS detection kit 2, we provide 1 ml of QB reagent and 1 ml of QB buffer (sufficient to prepare 100 ml of QUANTI-Blue™ Solution, a SEAP detection reagent). This should be sufficient to perform assays in approximately five 96-well plates (480 wells).

The LPS detection kit is highly sensitive and can detect as little as 0.01 EU/ml.

The use of some FBS might affect the functionality of HEK-Blue™-4 Cells as they may contain endotoxins which would interfere with your assay. Make sure the FBS used is endotoxin-free.
To avoid having endotoxin contaminants, we recommend to ask for manufacturer certification of endotoxin levels before purchasing the FBS to ensure it is endotoxin-free.

The cell line should be propagated upon receipt to make your frozen stocks for further use (no expiry date as the cells can be kept a very long time in liquid nitrogen).
Regarding the rest of the kit, it should remain stable for 1 year (providing that it is stored in the correct conditions)

There are 2 ways to avoid this nonspecific activation:
- You can either use our parental cell line as a negative control, the HEK-Blue™ Null1 cells (this cell line only expresses the NFkB inducible system). Therefore, any non-specific activation of NFkB will also be detected with the HEK-Blue™ Null1 cells.
- Otherwise, you can antagonize the TLR4 response with the Ultrapure LPS-RS which is an antagonist of LPS. If with this antagonist you eliminate the signal, this will then confirm that it is a specific TLR4 activation of your sample.
The reference of this antagonist is the following: LPS-RS ultrapure (#tlrl-prslps)

The cost of a customized cell line depends on multiple factors, including the complexity of genetic modifications, choice of the host cell line, licensing requirements, and additional validation steps. Just contact us, share your requirements, and we’ll provide a personalized quote.

The timeline varies depending on the complexity of the project and requests; it also depends on the cell line, growth characteristics, and culturing requirements. Typically, it takes our experts between 20 to 25 weeks, including cloning, selection, validation steps, reporting, and delivery.

Yes, we offer expedited development options to shorten the timeline where possible. Contact us to discuss your specific requirements.

Absolutely. You can choose multiple clones. For your convenience and scalability, we offer cell banking services, allowing for the production and delivery of multiple vials of your chosen clone(s) upon request.

Absolutely. We perform thorough QC testing, including sterility, mycoplasma detection, stability (up to 10 passages), expression validation, and functional assays to ensure high-quality results.

We perform stability tests to guarantee stability for up to 10 passages.

Our tech support team can advise on the best host cell line based on your research application, whether you need an immune cell model, adherent cells, or suspension culture. We work with a wide range of mammalian cell lines, including HEK293, THP-1, Jurkat, A549, Raji, RAW-264, and B16 cell lines.

No. To streamline your workflow we include complimentary samples of essential assay reagents, such as suitable selection antibiotics and reporter detection reagents with your final product delivery.

The cell-based data provides a more holistic understanding of the biological activity and the resulting cellular responses. This allows more informed decisions to be made about which compounds should enter therapeutic development.

Absolutely. We offer custom screening options for specialized research requirements. Whether you need unique PRR pathways or specific concentrations, our team can tailor the assay to meet your study’s needs.

Yes, of course! Our PRR Screening Service is designed to assess the potential activity of your compounds or extracts on various PRRs, providing valuable insights into their immunomodulatory effects.

Absolutely. InvivoGen offers many reporter cell lines suitable for screening lead compounds. From cytokine signaling to immune checkpoints, our team can tailor the assay to meet your study’s needs.

The price depends on many factors, such as the screening level, number of compounds and cell lines, concentration ranges, and grade of customization. The indicated prices (see above) cover:

- Compound Profiling (Level 1): 1 compound at 1 concentration on our TLR panel (7 cell lines) + control cell line.

​- Compound Dose Response (Level 2): 1 compound at 5 concentrations on 1 PRR cell line + control cell line.

For more tailored screening options, contact us, share your requirements, and we’ll provide a personalized quote.

In our experience, a minimum of 7 concentrations be tested with a series of 1:3 dilutions to sufficiently calculate an EC50 or IC50.

With over 25 years of expertise, InvivoGen offers robust, sensitive assays and customizable options. Our reliable data analysis, confidentiality, and commitment to quality make us a trusted choice in immunology research services.

PRR profiling is critical in identifying compounds that influence immune responses, aiding in adjuvant and drug discovery for conditions like cancer, autoimmunity, and infectious diseases. This process accelerates early-stage drug development by providing reliable data on immune pathway modulation.

The cost depends on project complexity, plasmid design, backbone selection, sequence, and final DNA quantity. Just contact us with your project details for a personalized quote.

Project timelines vary depending on sequence complexity and requested quantities. Typically, your custom plasmid will be ready within 2 to 4 weeks from order confirmation.

Yes. Plasmids can be delivered either lyophilized/dry or in liquid format, in the concentration and buffer of your choice (e.g., PBS, TE buffer, nuclease-free water, or custom buffer).

Standard production range from 20 µg to 10 mg of plasmid DNA. Upon request, we can supply higher quantities to meet your experimental needs.

Yes. We provide full sequence verification of the entire plasmid using third-generation sequencing method, ensuring complete accuracy. A detailed plasmid map and the full verified sequence are provided upon delivery.

Every construct undergoes stringent quality control, including sequence confirmation, purity assessment, and integrity validation prior to shipment.

Absolutely. Our technical experts can assist you in choosing the optimal plasmid backbone and guide element selection based on your downstream application. Just reach out with your project details.

Depending on your assay, you may want to store the supernatants for later reading. We recommend storing the supernatant at 4°C for up to 48 hours and at -80°C for a longer period. Do not store at -20°C.

There are different possible explanations for low absorbance values:
- The LDH reaction time is too short. If so, do not use the optional Stop Solution and perform kinetic readings of LDH activity.
- The cell culture density is too low. If so, repeat the experiment to optimize cell number.
- The test compound may interfere with the LDH oxydo-reductive reaction (see FAQ above).
- Bubbles are present in the wells when performing the reading. Avoid bubble formation when pipetting and mixing.

One possible reason for the high variation in replicate values could be pipetting errors or insufficient mixing of the Reagent Mix Solution with supernatant samples. We recommend you repeat the experiment using another set of pipettes and/or mix the plate more thoroughly (avoiding bubble formation).

Since LDH-Blue™ is non-radioactive and uses absorbance-based detection, it can be multiplexed with fluorescence or luminescence-based assays from the same sample (with appropriate timing). For example, you can use LDH-Blue™ with InvivoGen's Lucia luciferase-based cellular assays: the HMGB1 release reporter cells THP1-HMGB1-Lucia™, the ferroptosis reporter cells HT1080-HMGB1-Lucia™, and the oxidative stress reporter cells HEK-Lucia-Star™ ARE.

To monitor cytotoxicity using InvivoGen's LDH-Blue™, you will need:
- test and control supernatants
- pipettes and pipette tips, including multi-channel pipette
- reagent reservoir
- flat-bottom transparent 96-well plates
- a spectrophotometer microplate reader capable of reading 650nm absorbance.

The kit components are shipped at room temperature. Store the Reagent Mix at -20 °C and protect it from light. Store the Assay Buffer, Lysis Buffer, and Stop Solution at +4 °C. When stored as directed, the kit is stable for up to 1 year from the date of receipt. Once resuspended, the Reagent Mix is stable for 1 week when stored at -20 °C.

LDH-Blue™ detects lytic cell death associated with plasma membrane disruption, including necroptosis, pyroptosis, ferroptosis, late apoptosis, and immune cell-mediated cytotoxicity. It is not specific to apoptotic pathways without membrane rupture.

LDH-Blue™ primarily detects cell death associated with membrane integrity loss, such as necrosis or late-stage apoptosis. For early apoptosis detection, complementary assays like Annexin V staining are recommended.

Absolutely. LDH-Blue™ is ideal for T cell, NK cell, or antibody-mediated cytotoxicity assays. Ensure proper controls for spontaneous and maximum release.

Yes, LDH-Blue™ is sensitive enough to detect LDH release from samples with low cell numbers, including 3D microtissues and primary cells. Its high sensitivity ensures accurate cytotoxicity assessment in various experimental setups.

Absolutely. LDH-Blue™ is designed for use with standard 96-well and 384-well plate formats, making it suitable for high-throughput screening of cytotoxicity in drug discovery and other large-scale studies. You must change the volumes of the supernatant and reagent mix solution (and stop solution) according to the plate format. For a 96-well plate, please see the technical datasheet.

Yes, LDH-Blue™ is optimized for use with serum-containing or serum-free media. Background levels may vary slightly with serum type.

Certain substances, such as high concentrations of reducing agents or components that affect enzyme activity, may interfere with the assay. It's advisable to include appropriate controls to account for potential interferences.