I use GMCSF induced monocyte/macrophage culture (from PMBC) on 7th day for the activation assay, but always doubt the number of cells present per well. For 24 well plate I add 0.5 to 1x106 PBMC (Mixture of lyphocytes, platelets and monoctyes) After 18-20 hrs adhesion, suspension cells are removed and adhered cells are incubated with GMCSF for 7 days. How does one know that all wells have uniform macrophage distribution?
I doubt that distribution is going to be uniform. You better go for CD14 selection, maturation for 6 or 7 days with GMCSF. Then you will recover adherent macrophages by scrapping and redistribute them equally after counting. If your lab can not afford CD14 selection then you have to scrap your cells after the adhesion step, count them and distribute them equally. Hope it helps.
We culture macrophages from PBMC in PFTE coated bags, then plate them out for stimulation or whatever else. There is a paper with a few more details here http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3397322/. It gets really good yields and you can plate them out at known concentrations.
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