How much digestion mixture is needed and what are the exact amounts of collagenase P and dispase II needed?

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Alejo Cheung
Jul 17, 2023
protocol Protocol: Isolation, Purification and Characterization of Exosomes from Fibroblast Cultures of Skeletal Muscle

I’m trying to isolate fibroblasts from mouse skeletal muscle recently by following this protocol. Upon reading the procedures, I was confused with some of the details. For example, in step B7, you recommend 3.5x volume/mg tissue. Considering the GA tissue that I got weights about 100mg, Read more Read less more less

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Diantha van de Vlekkert Author Answered Jul 18, 2023

Genetics, St Jude Children's Research Hospital, United States

Dear Alejo,


Regarding your questions, Line B7 is 3.5x volume/mg in microliters, not milliliters.


You are correct with the calculations, for this protocol keep the final concentrations of 5mg/ml Col P and 1.2U/ml of dispase II. In addition, the digestion of the muscle will work also with half the concentration of col P, depending on the age of the mice and mouse model used. The time of digestion can be extended with an additional 15 min if needed.

I hope this answers your questions.

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