THP1 monocytes, despite of them being a suspension culture are getting adhered (all the cells) to the dish during serum starvation (at least 45mins to 1hr in basal RPMI). They are adhering so strong that I have to lift them off forcefully using a pipette by flushing with the medium. There is no such issue when I treat the This only happens during serum starvation and not with the normal culture (in 10% FBS + RPMI). I usually seed 5-10 lakh cells in 2-3mL in a 60mm dish for serum starvation.
Our lab performs THP-1 differentiation for specific experiments, using a protocol based on two papers: low dose PMA (5 ng/mL), 3 days (with or without rest for another 5 days; rest means removal of PMA and leaving cells in normal medium).
http://www.ncbi.nlm.nih.gov/pubmed/17334670
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0008668
In differentiation by PMA (3 days) followed by rest (5 days), the number of undifferentiated cells gradually increase over time. Undifferentiated cells resume the roundish, nonadherent properties of THP-1 monocytes.
However, whether such "regressive" behavior means the cells revert to exactly the original state before differentiation CANNOT be assumed (and should not be assumed).
***
beta-mercaptoethanol supposedly scavenges off some endogenous ROS (e.g. H2O2) and helps to keep THP-1 cells from producing excessive ROS and depletes its own antixodant reserve (e.g. GSH) prematurely. But there are alternative explanations.
https://www.researchgate.net/publication/231405783_Kinetics_and_mechanism_of_the_oxidation_of_2-mercaptoethanol_by_hydrogen_peroxide_in_aqueous_solution
Article Kinetics and Mechanism of the Oxidation of 2-Mercaptoethanol...
It's interesting observation.
I would speculate that the cells have been partially differentiated.
BD Biosciences published this manual for assessing THP-1 differentiation markers.
The best known conventional differentiation inducer for THP-1 is low dose PMA, but it takes days for full differentiation.
https://www.bdbiosciences.com/documents/BD_Multicolor_MonocyteMacrophageDiff_AppNote.pdf
many cell types (e.g. some stem cells, neuroblastoma cells) have been reported to be differentiated in serum-starved conditions.
Probably stress induced. Production of ROS and then differentiation of the cells. Try using B-Mercaptoethanol in your media (it's a free radical scavenger and stops differentiation).
Know this from experience: have cultured THP-1 cells.
I suppose you are culturing them in basal medium-0% FBS.
try giving the serum starvation in 1% serum medium.
(It could also be so that the medium with very less viscosity due to the lack of FBS might be leading to the settling of cells and eventually adhesion.)
Also culture them in dishes of larger space (60 mm dishes or T25 flasks)
Add more volume of medium (5-6 ml)
I agree with Fiona's suggestion. Consider adding 50 uM beta-mercaptoethanol, which is regularly used in THP-1 culture in many papers, anyways.
See: https://www.researchgate.net/post/How_to_Culture_THP1
Thank you, everyone. So, my cells seem to be partially differentiated. Would beta-mercaptoethanol help them revert to the normal condition or would it just work with the freshly revived ones?
Also, I have another question. If I now freeze these partially differentiated cells and revive them again after some days, is there any chance that they would revert?
Our lab performs THP-1 differentiation for specific experiments, using a protocol based on two papers: low dose PMA (5 ng/mL), 3 days (with or without rest for another 5 days; rest means removal of PMA and leaving cells in normal medium).
http://www.ncbi.nlm.nih.gov/pubmed/17334670
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0008668
In differentiation by PMA (3 days) followed by rest (5 days), the number of undifferentiated cells gradually increase over time. Undifferentiated cells resume the roundish, nonadherent properties of THP-1 monocytes.
However, whether such "regressive" behavior means the cells revert to exactly the original state before differentiation CANNOT be assumed (and should not be assumed).
***
beta-mercaptoethanol supposedly scavenges off some endogenous ROS (e.g. H2O2) and helps to keep THP-1 cells from producing excessive ROS and depletes its own antixodant reserve (e.g. GSH) prematurely. But there are alternative explanations.
https://www.researchgate.net/publication/231405783_Kinetics_and_mechanism_of_the_oxidation_of_2-mercaptoethanol_by_hydrogen_peroxide_in_aqueous_solution
Article Kinetics and Mechanism of the Oxidation of 2-Mercaptoethanol...
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