flow cytometry problem - (Apr/17/2006 )
I am trying to do some simple facs analysis to check which markers are present in my cell population. Can anybody suggest a protocol? I tried one and it didn't seem to work. The facs technician told me that my cells looked weird and that might be the reason I didn't get staining. I should have gotten a round pattern of scatter plot. Instead, I got a liner pattern in the plot... Any help?
My advice would be to try again with fresh cells and ask the Facs tech for help. What cell type were they? Were the cells fixed or viable? How soon after harvesting the cells did you go onto the flow cytometer? If you going to leave the cells overnight it's worth fixing them with paraformaldehyde.
All the best,
you could try simply staining your cells with Propidium Iodide first as a control - that way you'll know what they look like normally and then you can try out your markers......
I thinks its very simple only things you keeps in the mind if you disred to working on the intact cells keeps all working solution at isotonic forms otherwise its lyise your cells sample.
For example you fist time use PI staining of cells genomic DNA you detached cells if they are adherent and removed supernatent media by spining its. dispersed in the 1X PBS during whole procedure during fixating with 70% ethanol and finally stained with PI. after staining you directally analysis cells on the flow cytometer.For further quari you can contact me by this forum or by mailing at email@example.com
all the best.