Hello,
I'm newly working with THP-1 cells. I treated my cells with cell culture media containing 100 ng/mL of PMA and seeded them at a density of 3x10^5 cells/mL in a 24-wells plate. After 24h incubation with PMA, I checked the cells under the microscope, and the majority of them seemed to be attached to the plate. However, after washing with PBS, the cells got washed out. This is the 3rd time I face this issue and I cannot figure out why. I would appreciate any suggestions/tips on what I might be doing wrong. Thanks in advance!
I can give you a couple of ideas:
1) Maybe you are not washing your cells carefully enough, THP-1 cells are very delicate, if you pour PBS directly over them, they are gonna detach. Pour the PBS (and the medium too) over de walls of the well.
And do not shake them nor stir them (Just joking).
2) If I am no wrong, PMA 100 ng/ml it´s too much. I don´t know, but maybe your cells are dying and for that reason they are detaching. Examine if your cells are alive, if the concentration of PMA is adequate and if your cells do not lack any nutrients.
3) Although the cell concentration seems fine, maybe is too low. 5x10^5 cells/ml seems to be the ideal.
For what I see, the problem is the handling, just be carefull. but in your free time you should study why are you using the PMA concentration that you have described.
I agree with Pablo Veloso. We must wash carefully. I washed uncarefully once and I lost my THP-1 cells. Also, I used 50ng/ml of PMA to 5x10^6 cells in a 24 well plate, so maybe you need to increase
Thank you Pablo Veloso and Sabrina Maciel for your valuable suggestions.
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