I've been playing with flow cytometry and some J774 cells for a while but am a little confused by the FSC v SSC plots I'm seeing. I would really appreaciate it if anyone who uses these cells for flow can give me some advice!
I have experience growing and doing cytotoxicity and various other assays with J774 cells and I realise that they are extreamly sticky and grow like made! To reduce their grown I have reduced the FBS to 5%, and to unstick them I have found the best way is to pipette (w/p1000) and re-use the flasks for <10 passages as they seem to detach better when their home 'isn't new', as such
I have run them on the flow cytometer with PBS, with PBS+BSA, with media; after scrapping, after pipetting, after using Sigma cell uptake solution; after fixing and live .... regardless I always seem to get 2 populations of cells (see attachment), one that I have called 'P1' (red), and then the other stuff which looks like debris.
I guess my cells are 'P1', but is it normal for them to be so big ? (my FSC voltage is 4, and the max. is 1000!!), and I only just fit them onto the plot. And why so much debris ? Is this normal for J774s ? I realise I can set my FSC threshold higher (for example 1000 rather than 200) and then this would "remove" my debris, albeit only visually, but is it normal ?
Any advice would be greatly appreciated !!
Eskerrik asko, Moo