I am interested in characterizing surface markers related to epithelial/mesenchymal differentiation to characterize EMT via flow cytometry. The issue is that trypsin destroys the binding capacity of most antibodies for E-cadherin and N-cadherin. I am aware of using EDTA to dissociate cells, however, my cells are strongly adherent. Are there any other means of dissociating cells that I am not aware of? Alternatively, can anyone recommend antibody clones that have epitopes which persist following trypsinization?
You can use police man to scrape the cells in the flask
or Use cell disassociation medium
HI Adam,
The best method for these experiments is to use Nunc Up-Cell dishes, coated with a temperature reactive polymer, cell adhere at 37"c and float loose at room temp!! Amazing product, BUT each 90mm dish or 6 well plates costs about $40!!!!! I use them for critical experiments. the routine way we do this on my lab is to trypsinize the cells, then put them back into a flask on a rocking platform in the incubator so they can not re-adhere. We usually do this before going home and let them rock overnight. Next morning we do the staining and flow analysis or sorting. I can recommend the exact rocker we use if you want that info. Please see our paper "Expression of pluripotent stem cell reprogramming factors by prostate tumor initiating cells" the PDF is available on this site.
E Cadherin is probably more sensitive than N Cadherin to trypsin, but both are significantly removed and flow to measure them is totally unreliable directly after tripsin treatment. They are also damaged by accutase in my hands.
Kind regards,
Steve
Try collagenase type 4 from Worthington. Type 4 is designed to be especially low in tryptic activity to limit damage to membrane proteins and receptors. Good luck
Adam, worthington has a nice dissociation guide. I agree with Krish that collagenase 4 should be the first try.
- Badges
- Science method