I placed a sample of my cells in a flow cytometer, and I saw a monocyte population, but I also saw another cell population that were smaller than monocytes but more granular than them also. Wondering what that could be from the u937 cell line?
I think the cells you have are not in good condition and some of them are apoptotic cells (early stage). Often these cells are around 30-40% smaller. Another suggestion is, it could be a contamination. My recommendation is just discard these cells and start with a new cultivation. According to the granular visualization in U937 cells are easy detected during differentiation. These monocytes have the potential of differentiating into either macrophages or dendritic cells. When these cells are stimulated with Phyco-CM , PMA (TPA), RA thereafter the granular will be detectable.
I agree, the small cells are definetely apoptotic or dead. Exclude them from your cytometric analysis by propidium iodide (or any other dye for dead cells). I also agree that checking their identity by specific surface markers (CD14-, CD15+, CD33+, CD68+) or their capacity to differentiate into an adherent cell type is the best you can do. As for your culture condition, a HEPES-buffered basal medium does better in supporting optimum culture conditions than a carbonate-buffered one.
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