What kind of enzymatic digestion should I use and for how long ? trypsin or collagenase
Are you sorting based on an external marker? If so, don't use trypsin because you will cleave off your marker. You will likely have to use collagenase and EDTA.
Actually not, I am sorting based on the vital dye named conA that binds to glycoproteins containing α-D-mannose, α-D-glucos ( internally) but I am planning to use surface marker antibodies for some other type of cells. Do you have a protocol that I can follow for collagenase ( what concentration and which kind and how long to incubate my tissue with collagenase)? Thanks for the suggestion.
Here is the protocol I use for flow cytometry on mouse tumors:
1) (TUMOR) cut up with forceps in 24 well dish with 500uL BSS wash
a. Add 500uL 2X collagenase H (4ug/mL)
b. Incubate at 37C for 1hr
2) Mash tissue through 100um Falcon yellow strainer using a 5mL syringe plunger
a. Rinse tissue through strainer into a 50mL conical with BSS wash. Discard fat and connective tissue.
Spin 400xg for 5 minutes
3) Stain as you normally would
4) Pass samples through 40um blue cap Falcon strainer immediately before flow (shoot the cells through using a p200 or p1000)
(TUMOR) you may have to pass this though a yellow 100um strainer again before it will go through the blue 40um cap
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