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|250 µL of the diluted Cyto-ID® green stain solution was added to each sample and mixed well instead of 100ul|
|Incubate for 30 minutes at 37°C in the dark|
|Incase of Low CYTO-ID Green dye staining in all treatments, increase the reagent concentration (500X dilution of the dye is recommended) and the incubation time.
Incase of High CYTO-ID Green staining, it could be that the cell culture medium was depleted of nutrients and so change media 4-8 hours before the experiment
|Lyse cells in pre-cooled RIPA lysis buffer containing 50 mM Tris-HCl (pH 7.4) 150 mM NaCl, 1 mM dithiothreitol (DTT), 0.25% sodium deoxycholate, 0.1% NP-40, 1 mM phenylmethysulfonyl fluoride (PMSF), 50 mM sodium pyrophosphate, 1 mM Na3VO4, 1 mM NaF, 5 mM EDTA, 5 mM EGTA and a protease inhibitor cocktail.|
|Dilute primary Ab at 1:1000 and incubate overnight at 4ºC|