Hi Elliot,

FSC and SSC in FACS mainly reflects cell size and granularity. To be sure about the location of two distinct cell populations, you should run them separately. Then only you can confirm whether that low FSC/ low SSC population is cell debris or actual cells of small size and low granularity.

Secondly from the picture it seems that after 1 min of co-culture only few cells are +ve for the staining, but after 4h more cells are +ve for the stain. It appears that the comparatively low SSC population is your target population and it is not cell debris. You may try the same staining with FSC on X axis. That might give you a better result. Answer of the first question would solve the second one also as both the cell population seems to be same.

Hopefully, it would help you......

As you have your FSC and SSC shown, I usually do not include anything below 30k to be an actual cell.  This is from multiple experiments with many primary cell types.  You should include some sort of "dump" staining that will remove these events for you.  A dump is using antibodies you know your cells do not possess (if you are using T cells, then CD19, CD20 for B cells, Ly6G for neutrophils) tagged with a fluorochrome you are not using and then you remove those "dump+" events as trash or not cells of interest.  Another thing that should inform your analysis is you should at least know what the relative amounts of each cell type are.  That should match what you see at least at the 1 minute time point in your flow data.  If it is a 50/50 mix the flow data should be 50/50.  Also, if you can induce death/apoptosis in a tube with target cells alone, that might inform what those cells look like as they die by flow.

Thank you Steven for the reply. I will try your Dump staining advice and will look at my relative amounts. I do have tubes of target cells that have been induced to die that are stained with annexin V, so  I will look at that as well. Thanks again for the quick reply

Elliot