questions about isolation of epithelial cells

Hi ZHANG QINGYI,

1. It’s a good point. Actually, the usage of either 50ml or 15ml tubes is optional. we do think that in case of inflammation (where the epithelial fraction is minimal) it’s better to use 15ml tube in the step C2h (for a better visualization of the pellet).

                  g. Add 1 mL of FBS into the 5 mL of TrypLE to stop the reaction.

                  h. Filter through a 40 μm strainer in a 50 mL conical tube. Add epithelial wash buffer up to 50 mL. (In case where a 15ml tube is used, after filtration, transfer the contents to a 15ml tube and add epithelial wash buffer up to a total volume of 15ml).

• For the DNase I: the concentration of DNase I is mentioned in this step:

c. Resuspend the crypt pellet in 5 mL of pre-warmed TrypLE containing DNase I (100 μg/mL).

If your stock is 100mg/10ml (=10mg/ml), then for 5ml of TrypLE, you need to add 50 μL of DNase I (dilution 1/100 to get the concentration of 100 μg/mL).