THP1 Cell lines

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.