One of my cell lines shows up as positive in APC channel without any APC conjugated antibody by flow.
I only have two colors which was stained separately so none of the samples have both APC and FITC.
I used another cell line to set up compensation and it looks OK.
Can this still be compensation? high auto-fluorescence for this particular cell line? Has anyone seen this?
Thank you!
do you have a fully unstained sample (no FITC or APC), using this cell line? what did it look like? It's best to use the same cell line, unstained, to set voltages - autofluorescence isn't uncommon. what cell line is it, out of interest, and what cell line did you use to compensate?
First of all, I agree that you need an unstained sample with the same cell line. Secondly, how do these cells with fake APC positivity appear?I mean the FSC and the SSC as compared to the other cell line. Moreover, did you add any compound to discriminate between apoptotic-necrotic events and living cells?
I suggest you to perform the staining with a completely unstained sample of the experimental cell line and to add dyes such as DAPI or PI for eliminating dead events from the analysis, that in turn appears a little more autofluorescent as compared to living unstained events.
Hi Everyone and Yumi Nakayama , did you ever work this out? I am seeing APC-like emissions from unstained HL-60 cells. Staining with our APC-conjugated antibody is brighter than the background autofluorescence and shifts the population to the right, but I am very confused as to where this comes from (and it's not just a little bit, up to 4th decade). I backgated on SSC/FWS and most cells homogenously have this autofluorescence, so I cannot gate them out of the analysis. Where does this come from?? Thanks in advance
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