TITLE: Adult mouse skin dissociation protocol (on ice)
AUTHORS: Andrew Potter

[DESCRIPTION]
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. 

[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)
 

[STEPS]
SECTION: Isolating tissue
1. After euthanizing mouse, remove hair using Nair: dab with Nair, wait 30 secs, wipe with wet paper towel.
SECTION: Isolating tissue
2. Isolate tissue and place in ice-cold hypothermosol.
SECTION: Isolating tissue
3. Scrape off underlying layer of fatty / connective tissue using scalpel before proceeding.
SECTION: Isolating tissue
4. 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.
4 min

SECTION: 1st digest layer
5. Place 23 mg minced tissue into 1 mL B. Lich enzyme cocktail. Incubate on ice.
23 mg


SECTION: 1st digest layer
6. Shake every min; triturate 10x every 2 min with p1000 w/tip cut (start triturating at 2 min) for 20 min.
20 min
1 min2 min



SECTION: 1st digest layer
7. 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.
2 min8 min


SECTION: 1st digest layer
8. Transfer back to 1.5 mL tube using 1 mL serological pipet. Mix thoroughly and allow to settle on ice 2 min.
2 min

SECTION: 1st digest layer
9. 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 µL5 mL


SECTION: 2nd digest layer
10. Add additional 1 mL b. Lich enzyme mix to residual tissue chunks.
1 mL

SECTION: 2nd digest layer
11. Triturate 10x every 2 min, shake every min while incubating on ice for 20 additional mins. (50 min. total digest time).

20 min
2 min1 min






SECTION: 2nd digest layer
12. 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

SECTION: Preparing cells for single cell analysis
13. 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.
5 min100 µL


SECTION: Preparing cells for single cell analysis
14. Adjust concentration to 1,000 cells/µL for Chromium or 100 cells/µL for DropSeq.