I am looking at PAR expression in our particular cell line. Unfortunately though, Trypsin, which is generally used for cell for cell detachment during passaging etc. is well known to activated PAR-2. Does anyone working with PARs have any suggestions for a substitute? Keeping in mind I am also looking at the other three PARs. I know of Accutase, but there isn't much info about it's mechanism of action. EDTA may also not be ideal given it works by chelating Ca2+ ions, which may (?) also affect PAR signalling.
Hi Tom,
you could try EDTA (10 mM in PBS) for dissociation and substitute with Ca2+ afterwards; another option for dissociation would be urea (0,5M).
Best regards,
Christian
While working with adherent cell lines and phospholipase , I have used "rubber policeman" scanning of cells from the substrate at 4C (plates were kept on ice". Trephination some times activate phospholipase and interfere with lipid studies. I have also used cold methanol during the scrapping. I am not sure if these treatments would interfere with your down stream analysis
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