Mixed glia cells were isolated from brain of P3 and P5 mice pups (differently isolated), cultured in Poly L-lysine coated T25 flasks (3 brains per flask) for 14 days (at full confluency). Microglia are harvested by rigorous banging and plated in 5cm uncoated bacteriological plates, incubated for 1hr, then add MCSF-treated media. After 7 days in MCSF media, they proliferate less, look unhealthy and dying although showed few processes.

What could be the problem? Advice are appreciated as I am new to primary culture. The cells are meant to be used for phagocytotic assay.

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