Feb 01, 2023
Version 3
Adult mouse skin dissociation protocol (on ice) V.3
- 1CCHMC
- Human Cell Atlas Method Development Community
Protocol Citation: Andrew Potter 2023. Adult mouse skin dissociation protocol (on ice). protocols.io https://dx.doi.org/10.17504/protocols.io.n92ld391og5b/v3Version created by Andrew Potter
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: February 01, 2023
Last Modified: February 01, 2023
Protocol Integer ID: 76216
Keywords: CAP, skin, dissociation, single cell, adult mouse skin dissociation protocol, mouse skin, bacillus licheniformis protease cocktail, ice, layers of digestion, protease, mouse, digestion, cell, protocol
Abstract
This protocol was developed to dissociate adult (8-10 wk) mouse skin "on ice". It utilizes two layers of digestion with a Bacillus licheniformis protease cocktail, combined with mechanical disruption from a dounce homogenizer. The cell yield is 3000 cells/mg.
Attachments
Guidelines
Bacillus Licheniformis Enzyme Mix (1 mL per 23 mg tissue):
100 μL b. lich 100 mg/mL (10 mg/mL final) (Sigma, P5380)
1 μL 0.5 M EDTA (Sigma, A8806)
899 μL DPBS (no Ca, Mg) ThermoFisher (cat. #14190)
Preparing enzymes:
The enzyme is made up in DPBS (#14190). It is aliquoted and stored at -80 °C at 100 mg/mL in 100 μL aliquots..
Reagents
Enzymes and BSA are made up in DPBS (no Ca, no Mg) from Thermo Fisher (14190).
Bovine Serum Albumin - Sigma (A8806).
Hypothermosol FRS
Required supplies:
2 mL dounce homogenizer – Bellco (1984-10002)
Centrifuge for 1.5 mL, 15 mL conicals
Pipettes and pipet tips
15 ml Conicals (MLS)
1.5 mL tubes (MLS)
30 µM filters - Miltenyi (130-098-458)
Petri dishes (MLS)
Razor blades (MLS)
Ice bucket w/ice
Hemocytometers - InCyto Neubauer Improved (DHC-NO1-5)
Isolating tissue
After euthanizing mouse, remove hair using Nair: dab with Nair, wait 30 secs, wipe with wet paper towel.
Isolate tissue and place in ice-cold hypothermosol.
Scrape off underlying layer of fatty / connective tissue using scalpel before proceeding.
Mince skin tissue thoroughly on petri dish on ice for 3-4 min on ice into 1-mm3 pieces using razor blade while manipulating tissue with forceps – you will need to use grinding motion and vigorously break up tissue.
00:04:00 mincing
1st digest layer
Place 23 mg minced tissue into 1 mL B. Lich enzyme cocktail. Incubate on ice.
23 mg minced tissue
Shake every min; triturate 10x every 2 min with p1000 w/tip cut (start triturating at 2 min) for 20 min.
00:20:00 digest
00:01:00 shake 00:02:00 triturate 10X
After 20 mins of triturating on ice, use pipet to transfer digest mix to 2 mL dounce homogenizer. Use 10 strokes of Pestle A every 2 min (4 series total, 8 min). Digest mix should become turbid.
00:02:00 dounce homogenize 00:08:00 digest using dounce
Transfer back to 1.5 mL tube using 1 mL serological pipet. Mix thoroughly and allow to settle on ice 2 min.
00:02:00 settle on ice
Save 70% (700 µL) of supernatant, leaving chunks at the bottom of the tube; apply to 30 µM filter on 15 mL conical. Rinse filter w/5 mL ice-cold PBS/BSA 0.04%. Save flow through on ice and keep filter on tube for 2nd layer.
700 µL save supernatant 5 mL rinse filter w/ice-cold PBS/BSA 0.04%
2nd digest layer
Add additional 1 mL b. Lich enzyme mix to residual tissue chunks.
1 mL b. lich enzyme mix
Triturate 10x every 2 min, shake every min while incubating on ice for 20 additional mins. (50 min. total digest time).
00:20:00 additional digest time
00:02:00 triturate 10x 00:01:00 shake every min.
Transfer entire volume to same 30 µM filter on 15 mL conical. Rinse with additional 5 mL ice-cold PBS/BA 0.04%.
5 mL ice-cold PBS/BSA 0.04%
Preparing cells for single cell analysis
Centrifuge at 300 g for 5 min at 4 °C. Remove supernatant & re-suspend in 100 µL PBS/BSA 0.04%. Examine using hemocytometer with trypan blue.
00:05:00 centrifuge at 300 g 100 µL re-suspend in ice-cold PBS/BSA 0.04%
Adjust concentration to 1,000 cells/µL for Chromium or 100 cells/µL for DropSeq.