any experts in the field of generating single-cell suspension from adult mouse skin?
I can not use any protocol with Trypsin, too harsh.
Tried both Liberase DL/TL digestion enzymes from 60 ug/ml - 300 ug/ml, following published protocols.. they make it sound easy, but I found the mouse skin is too tough to digest compared to the mouse ear/colons, that I have previously worked with.
Predigesting buffer: HBSS w/o Ca+/Mg+
5mM EDTA,
10mM HEPES
10% FCS
Digestion buffer IMDM
60 - 300 ug/ml Liberase TL
1%FCS
1000 U/ml DNAse type I
Pre-digest:
1) 30min, 37℃ incubation @ 150 rpm shaker, vortex 10sec, at end of the incubation
2) Filter through 70 µm strainer, collect tissue, discard flow though
3) Place skin into a 50 ml conical tube containing 10 ml RT Pre-digest buffer
4) Mince the tissues
5) 30min, 37℃ incubation @ 150 rpm shaker, vortex 10sec, at end of the incubation
Librase Digestion
1) Filter through 70 µm strainer, collect tissues
2) 10 ml Digestion buffer, 30min, 37℃ incubation @ 100 rpm shaker, vortex 10sec, at end of the incubation
3) Filter through 70 µm strainer, wash with cold PBS, keep supernatant @ 4℃
4) collect tissues for repeat digestion 2)
Filter through 70 µm strainer, wash with PBS, pool samples supernatant 3), spin @ 400x g, 4℃ for 10 min
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