how do i do flow cytometry for adherent cells? - (Jul/30/2007 )
Hello all. I am using siRNA to knockdown lamin proteins in HeLa cells after which I will add FITC antibodies and then follow with flow cytometry. Basically, I'm not sure what is the proper protocol for doing flow cytometry with adherent cells. Should I detach the HeLa cells before fixation,permeabilization,adding antibodies. Or, can I do all that while the cells are attached and then detach them somehow right before flow cyt?
I have tried the later and detach using trypsin but somehow trypsin seems to be chewing up all of the cells and oddly enough, i don't even see any cell debris. It's like the cells just disappear after I add trypsin. If I add a smaller concentration of typsin, the cells will not detach but those that do detach will also get chewed up. Is there a better way to do this? Thanks so much!
well i've not done too much of that kind of assays.
I would go for treatment on adherent cells and then go for cytometry.
you will be more able to control the confluency, and will not be disturbed by clumps of cells that may interere with treatment
Ironically I want to do almost exactly the same thing. HeLa cells and all, unfortunately from what I can find this will not work. I am told fluorescence microscopy is the best. Problems are: trypsin digests cells up, cant detach cells from one another reliably so clumps go through the flow instead of single cells (you can't fix cells first for that same reason), the cells stick to the tubes used for flow cytometry... Probably more, so I am thinking it may be impossible to do adherent cells by flow. I really really really want to be wrong, so if anyone knows a trick or if you find a way I would LOVE to know!!
I was replying at the same time as fred_33 and his reply does suggest a possibility, perhaps by culturing very subconfluent cultures you can fix the cells on the plate then use a cell scraper to remove into a tube for flow??? I think I may try that... if you get a chance to try and it works PLEASE let me know...
Good Luck again!
i've done co transfection with plasmids which have included a GFP.
So i trypsinise the cells as usual, helping by pipetting up and down with medium at end of trypsinisation process.
I collec all in tubes,and just before analysis in the cytometer, i use a 1000µl pipett and pipett up and down the cells for 10times rouglhy.
The result was no clumps.
I did that on facs aria with 100µm size limit i think (the person who deals with cytometer is not available at time srry )
I never use HeLa cells, I only use MCF7 cell lines.
After trypsin, use a two ml pipette to break up the clumps in the cell suspension, by going up and down three times. Most of the clumps will be dissociated into single cells.
Then do fixation,permeabilization,adding antibodies.
Hope this may help.