Hello everyone
I am differentiating MDM form human PBMC using M-CSF for 6 days. My problem is that when I read my macrophages on the cytoflex for flow cytometry, they do not fit the forward scatter/ side scatter scale and generally look like a weird S shape. They look slightly better on flowjo but they still do not look like a neat and tidy population. When I stain this population with AnnexinV, I can see that only about 10% of them are expressing AnnexinV, meaning the majority of my cell population is most likely alive. Has anyone ever encountered this weird ffs/ssc plots and what can I do to fix them? I am attaching the pics of what they look like on the cytoflex and flowjo. Both are unstained populations. Thanks
If you add Propidium Iodide to your staining you will be able to find out about the early late apoptosis and necrosis. It looks like there are two populations with different granularity.
I think it's because of insufficient differentiation from monocytes to Macs. You may need to another 1 or 2 days to harvest the macrophages. The other reason may be the concentration of M-CSF is not high enough. Another thing, the gate only covers ~45% cells, which does not fit the live cell population (should be ~90% if you have 10% Annexin V+) covers. So you may need to adjust the voltage to cover most of cells. Good luck.
Adjust the voltage during acquisition to fit most cells in the axis range.
Maybe some of your cells outside the axis are doublets, remove them by excluding any cells out of the main population seen on a FSC-A/FSC-H graph.
As Louis-Charles recommended, just adjust the voltage. Compared to lymphocytes M-CSF differentiated macrophages are much much bigger.
After 6 days of M-CSF differentiation (50-100 ng/ml), most of your cells should be fully differentiated. I also observe two populations with different granularity.
Hello Bengisu,
The DENSITY plot looks nicely positioned. The tail(low FSC/SSC) is likely your apoptotic cells and should gate ~10%. The DOT plot does not appear to have the same sample profile - the detector is over voltaged on the SSC!
If these are the same sample, I would highly recommend you contact Beckman's technical support team. This does not look like the same sample!
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