Hi, I'm culturing THP-1 and induce them into macrophage. And I run into some issues:
I culture 2x10^5 cells / ml and seeding 2x10^6 cellsin a 6 well plate, treat 20ng / ml PMA for inducing them to macrophage.
I found that cells tend to cluster, and the induction fail (cells not adhering), I'm ont sure where may problems occurs, only have some speculations:
1. cell density is too high. But seniors in our lab think 2x10^6 is fine.
2. PMA stock failed after freeze and defreeze multiple times (does anyone encounter this issue?).
3. cells with unknown issues, since this batch is newly defreeze.
Can someone provide other possibility or any suggestions on culturing THP-1? Thinks!
Hello Yuhung Zheng
THP-1 cells are sensitive to cell density. The recommended seeding density is between 3-7 x 10^5 cells/ml. Generally, 5 x10^5 cells /ml is used.
If you have just thawed the cells recently, then you need to increase the FBS content to 20% for the first two to three passages, then you can switch over to 10% FBS for successive passaging.
You need to add 2- mercaptoethanol to a final concentration of 0.05 mM to the basal medium, RPMI-1640 containing 10% FBS.
Sometimes cells may clump. But if they are growing fine and have a normal morphology it should not be a problem. You need to remove dead cells if they are present as sometimes these dead cells could be the cause of clumping.
THP-1 cells grow better in conditioned media. Therefore, if necessary, when passaging cells, you can leave 1-2ml of old media and add fresh media to the culture. You should use fresh PMA stock when planning your experiment since PMA has been used several times. Also, optimization of PMA concentration and treatment time is essential.
Good Luck.
Hi, from my point of view, the cell density is too high. Adjust the concentration around 0.3-0.5 mill/ml, and seed 2 ml/well in a 6-well plate
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