Hi folks,
I am growing RAW 264.7 cells in 12 well plates, RPMI with FBS and PS.
When harvested and acquired cells in FACS, in few of the stimulated wells population is missing or second population positive for CD11b appears, but unstimulated has macrophages, can anyone suggest how to deal this? no trypsinization was performed just used scrapper,,
Hello!
1. Did you passed resulted cells via filter (cell stainer)?
First kind of stimulation looking like control cells, second possibly contain a lot of dead cells/apoptotic bodies, and the last possibly contain a lot of fused cells or cell aggregates (phagocyted?).
Alternatively try to inspect cell suspension under microscope to see changes in cell morphology.
Maybe trypsinization will not so bad for them. In our experience we've used cells after trypsinization to study CD11b/c expression.
Also you could find helpful treatment with 1 mM EDTA in PBS (5-15 min, 37 oC) before scrapping.
Hi Petrenyov
Thanks for your answer,
I didn't pass the cells through strainer,, morphology under microscope looked small and less elongated in unstimulated whereas stimulated cells are long, well elongated and adhered in plates,,,assumed scrapping should be fine, but also planning for trypsin treatment next time,,,
will try EDTA method and share the outcome,,
Growing RAW cells in DMEM is preferred? read that DMEM has high glucose concentration and down-regulates many markers in macrophages, is it true?
Thank you once again,,
Hey Santhi!
I have a similar question.
I cultured the cells in DMEM media and when I did just the flow analysis of raw264.7 without any treatment, I find that there are two distinct populations. Can you explain if you have same patterns..
Thank you!
Hi Abishai
Yes, I too observed 2 distinct population (kindly find the previous attached slide). Passing cells trough strainer worked for me but I didn't repeat them to check the reproducibility. were both the populations positive for your phenotype staining? if so it creates further bias, otherwise you can continue analysis on the positive population.
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