I need to run flow cytometry with endothelial cells. Maybe my cell culture is a bit over confluent. After detached, the cells with trypsin-EDTA are pipetted up and down so many times, and I still see a lot of cell aggregations. The clumpy cell culture may block the machine. So, I am wondering whether anybody else has run into a similar issue and has a solution for it?
Do not use trypsin. It damages primary endothelial cells causing nucleic acid release. You can treat with a DNase/RNase cocktail. Alternatively, use a non-trypsin based enzyme product such as Accutase.
Thanks, Michael. I forgot to mention that the endothelial cells I work with are not primary cells. It is a virus transformed cell line from ATCC (SVEC4-10 is an endothelial cell line derived by SV40 (strain 4A) transformation of endothelial cells from axillary lymph node vessels.). Detachment with trypsin-EDTA is recommended in the protocol.
Be s ure to wash the cells with Calcium/magnesium free buffer so as to limit trypsin inhibition. We use a 40micron filter to prevent "flow back-up". Good Luck1
Yeah, definitely wash with Ca/Mg free PBS before the trypsin/EDTA. Also, you could adjust the concentration of trypsin.
Finally, traditionally, cell clumps are dealt with via trituration, essentially pipetting them up and down several times -- which you're doing. Since it's not working, try a small gauge needle instead. Transfer the suspension into a sterile disposable syringe, cap with a 22g needle (don't go too small, since you can shear the cells apart that way) and gently expel the solution. Repeat a few times, as needed.
Yes, washing with Ca/Mg free PBS before trypsin/EDTA is required. I would also suggest to wash the trypsinized cells with PBS.
Finally, clump removal is an essential step for FACS analysis. Pipetting them up and down slowly for several times is the normal procedure. If it does'nt work well, switch to a sterile small gauge needle and repeat the procedure for 5-6 times (but slowly to avoid cell disruption). Hope this would work well.
Hi
How long are you incubating the cells in TE. In my experience, washing the cells with 0% media or PBS prior to TE treatment and then capturing cells using serum added media doesnot cause cell clumping. Incubate with TE only long enough to detach the cells (you can check in microscope). after capture, pipetting 2-3 times should give good cell suspension for FACS.
Hi Li
Agree with previous posts about washing with PBS.
But would suggest that if you are going to pass your cells through a syringe, don't go smaller than an 18G needle. For my cells, 3 passes will get rid of clumps, but with very minimal shearing effects (I found significant shearing with 22G, but I'm guessing it depends on your cell size).
I would also try to avoid any bubbling as cells caught out of the medium will behave differently.
Wash your cells following harvest with PBS lacking Ca2+ and Mg2+. This will help the cells to not clump up as much. You can also filter your samples using special flow tubes with filters. Make sure you vortex your samples well before putting them on the cytometer to run.
I agree that you need to wash your cells in Ca/Mg free PBS. But sometimes in experimental set up like transfected / virus infected / drug treated cells clumping of cells occurs and it is very hard to make single cell suspension even after vigorous flushing. I am having very bad personal experience in virus infected cells. You can follow following steps
After trypsin-EDTA treatment tap gently the disc/plate and observe under microscope for proper detachment of the cells from the surface, then add ice cold complete media (don't add PBS at this step, what I used to add initially) to shut-off the effect of trypsin-EDTA, followed by spin at 1800 rpm for 5 min at 4 dc and then use for Flow cytometry.
Note: If you are fixing cell in 70% alcohol prior to staining you should add alcohol slowly drop by drop with gentle vortexing in between.
Hope it will work
Tapas
In addition to the above, you could also try adding some EDTA (~2mM to 5mM) to the FACS running buffer which may help to prevent re-clumping of cells after they have been filtered.
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