General Cell lines 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.

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.

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.

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.

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
• Do not use trypsin (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.

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).

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.