Hi,
I need to perform a FACS experiment on strongly adherent cells (U2OS). Does anyone have experience in preparing these cells for FACS without using acute dissociation treatments such as trypsin? In my case, it would be crucial that the cell surface proteome remains intact as I intend to stain for a cell surface protein.
There are many different ways to dissociate your cells from the culture flask, including trypsin, using a cell scraper, EDTA and several other compounds, such as accutase, which have less severe effects on the cells than trypsin. I would test several methods to determine what would be best for your cell surface protein.
For some opinions on different methods of detaching cells:
https://lists.purdue.edu/pipermail/cytometry/2007-October/033669.html
Thanks guys for the answers! I now tried 10 mM EDTA in PBS and it works well in my hands.
I agree with Elsenoor from above. I use Accutase for both epithelial preps as well as macrophage preps. I think it works really well while preserving viability. Also, Accutase does not require serum deactivation (although I always wash the prep with relevant media, KSFM for epithelial cells and RPMI with 10% serum for macrophages).
There is also Accumax, which is effective at room temperature and I have found that it also works well although I do not really know the differences between the two besides that Accutase has some phenol red.
hello Joseph.. this is mubin, even i want to use FACS for ROS analysis in adherent epithelial cell line. Does accutase on its own produce ROS in cells? and do i have to wash the cells once I detach them from the plate surface? and also how do i make single cell suspensions as i read on the FACS website that we need single cell suspensions.. please suggest
thank you
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