
Detachment of very adherent cells for flow cytometry
#1
Posted 23 July 2009 - 04:40 AM
I'm having a bit of a problem with my cell line. We are using a new renal epithelial cell line which differentiate very nicely. We are trying to do some flow cytometry, but one of the side effects of the differentiation process is that the cells lay down a pretty impressive basement membrane. So much so that 10x Trypsin and Accutase really don't have an effect after 30 minutes or so at 37 degrees! Does anybody know of an alternative method (aside from scraping) to the two that I've mentioned to remove the cells from the plastic? Any advice would be very much appreciated!
Thanks,
Rob
#2
Posted 23 July 2009 - 05:18 AM
do you use trypsin with EDTA? from invitrogen you can get trypsin 0.25% plus EDTA (normally we use 0.05% plus EDTA).
#3
Posted 23 July 2009 - 05:29 AM
These cells are incredible, they are washed in PBS without Ca and Mg and our trypsin is 10x trypsin (i.e. 0.5%)! There is no EDTA in our 10x trypsin, but the Accutase does have EDTA and it doesn't work. Would pre-washing with EDTA, and then adding trypsin maybe help?you wash your cells with PBS w/o Ca and Mg before stripping? and it stills stick? wow.
do you use trypsin with EDTA? from invitrogen you can get trypsin 0.25% plus EDTA (normally we use 0.05% plus EDTA).
#4
Posted 23 July 2009 - 05:57 AM
Edited by cancergeek, 23 July 2009 - 05:58 AM.
#5
Posted 23 July 2009 - 06:24 AM
#6
Posted 23 July 2009 - 06:40 AM
#7
Posted 23 July 2009 - 07:04 AM
It does seem like a really good product, I was working with a polymer like that for my undergrad thesis and was surprised that there wasn't a million and one different uses for it. This one is very elegant indeed! I've ordered the free sample plates but I will definitely try adding EDTA to my trypsin to see whether that works. Thanks for the help guys/girls. I'll let you know how it goes.i would try to add EDTA with the trypsin, but the product cancergeek is talking about seems very interesting. I didn't know it. (but my cells are detaching with EDTA, or trypsin or accutase)
#8
Posted 24 July 2009 - 01:06 AM
However these plates are expensive and so what I do to detach my (very) adherent cells is remove the media, add 5mM EDTA for 15-20 mins, pipette up and down in each well and remove the EDTA and cells. Then add 10% FCS in PBS and wash each well again and add the wash to your cell mixture. This stops the cells from being so sticky. Then I just wash the EDTA off my cells. I had lots of problems detaching my cells and this method just pops the cells right off the plate.