I isolated CD3+ Cells from frozen PBMCs and then I seeded them in a 96-well plate in different conditions with some independent variables.
In this experiment, I need to incubate the plate for 7 days and then I do FACS analysis.
The problem is that after 1 week I lost around 70-80% of cells in each condition.
What should I do to lose a low number of cells during the incubation?
Hi!
How many cells did you seeded?
Did you added IL-2?
Could you elaborate the conditions you used and the concentration of each?
Hi Nam Tae Kim
Firstly, I isolate the CD3+ cells from PBMCs. Then I seed 200.000 cells/well.
In baseline condition only I have IL-2 and cell medium.
In the second and third condition I have , anti-CD8 and IL-2 and my independent variable.
Note: Before seeding I coat the plate (the wells that I need the activation of T cells) with 5 µg/ml anti-CD3 in PBS.
Nam Tae Kim
Also, another question, Should I change the medium of each well during g this 7days incubation?
if yes, how?
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