Effect of trypsin on cells in FACS - (Mar/08/2008 )
i use trypsin to detach cells from well plate in order to carry out FACS analysis. i m carrying out FACS to detect change in plasma membrane and also antibody binding. is it possible that trypsin generates change in cell morphology? is there any other option to detach cells effectively. (i do wash cells after treating with trypsin).
Morphology changes upon trypinization. But I regulary FACS aderent cells, so as long as you always treat them the same the morphology-changes between two experiments will be the same (I fix my cells after trypsinisation).
Apart from this: you need to be sure your that the epitope your antibody binds to is not getting chewed off by the trypsin.
You could scrape your cells, but that would cause stress maybe? another trick would be to detach your cells with trypsin and put them in polystyrene tubes for 2 hours (in growth medium, maybe more than 2 hours is possible with your cells) and then do the rest of your procedure.
Apart from this: you need to be sure your that the epitope your antibody binds to is not getting chewed off by the trypsin.
You could scrape your cells, but that would cause stress maybe? another trick would be to detach your cells with trypsin and put them in polystyrene tubes for 2 hours (in growth medium, maybe more than 2 hours is possible with your cells) and then do the rest of your procedure.
This helps, thanks Vairus.
Some time ago I'm studying reactive oxygen species (ROS) in epithelial cells by flow cytometry. The tripsinización and scrapping cause stress to the cells and control cells had very high levels of ROS. After trypsinization, we left cells in the incubator 2-3 hours before treatment to try that levels of ROS of the checks were baseline.