I am currently isolating bone marrow derived macrophages from femurs and plating them in 24-well plate (400k/ml in each well). I'm facing issues with poor attachment and low cell count. How should I troubleshoot this?
Hi Priyanka,
What is the composition of the media and what is the nature of the plate you are using?
We Use 20% HI-FBS supplemented, 30% L929 conditioned medium, 1% L-glutamine, 1% sodium pyruvate, and 1% penicillin/streptomycin in DMEM. From my experience, a Non-TC plate works best for BMDM. Do not disturb the plate until Day-3 (Count the day of the plate as Day-0). On Day-3 remove half of the media and add fresh complete BMDM media. Afterward, check the plate every other day. In my case, I found Day-7/8 best for experiment/freeze.
Alternatively, you can try differentiating cells in a 100 mm Petri dish (10 million per dish-Non-TC). Allow them to grow till Day 7/8 and lift the cells using a Cell stripper. Count the cells and plate macrophages ( approx. 1M/6 well, 0.5M/12 well..) for downstream application in regular media (you do not need L929 conditioned media at this stage).
Hope this will help.
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