TITLE: Adult mouse lung cell dissociation (on ice)
AUTHORS: Andrew Potter

[DESCRIPTION]
This protocol was used to dissociate adult (8-10 wk) mouse lung tissue. The entire procedure is carried out on ice (to reduce artifact gene expression changes) and takes about half an hour. The yield was 16,240 non-RBC/mg tissue with 87% viability. 

[BEFORE_START]
-Prepare enzyme mixes and leave on ice.
-Cool centrifuges to 4 °C.

[GUIDELINES]
Enzyme Mixes
 
Coll. A/Elastase/Dispase Enzyme Mix (1.5 mL --> save 0.5 mL of coll./elastase/dispase mix in separate 1.5 mL tube. )
90 µL Collagenase A 100 mg/mL – 6 mg/mL final (Sigma, 10103578001)
150 µL elastase 43 u/mL - 4.3 u/mL final (Worthington, LS002292)
150 µL Dispase 90 u/mL – 9 u/mL final (Worthington, LS02100)
7.5 µL 1 M CaCl2 – 5 mM final
7.5 µL DNAse (125 U/mL) (Applichem, A3778)
1095 µL PBS
  
B. Lich enzyme mix (1 mL)
899 µL DPBS (no Ca, no Mg) (Thermo Fisher, 14190144)
1 µL 0.5 M EDTA (MLS)
100 µL Bacillus Licheniformis 100 mg/mL - 10 mg/mL final conc. (Sigma, P5380)


Preparing stock aliquots of reagents:
Enzyme stocks are made up in DPBS. I make up 100 mg/mL stock of Collagenase A and Bacillus Licheniformis. For elastase, I prepare 43 u/mL stock, and for dispase a 90 u/mL stock.  Enzymes mixes are aliquoted in 150 µL (elastase, depase) or 100 µL (Collagenase A, Bacillus Licheniformis) and stored at -80 ºC. For the CaCl2, I prepare a 1 M stock and autoclave. The DNAse is diluted in DPBS, aliquoted and stored at -80 ºC.
 

Required reagents: 
Red Blood Cell Lysis Buffer (Sigma, R7757) - store at RT
Trypan blue solution, 0.4% (ThermoFisher, 15250061)
BSA (Sigma, A8806)
  
(Optional) Dead Cell Removal Kit: 
EasySep dead cell removal (Annexin V) kit (cat. #17899)
EasySep Magnet (cat. #18000)
  
Required Equipment & Consumables:
Centrifuges for 1.5 mL and 15 mL conicals (MLS)
Pipettes and pipet tips (MLS)
15, 50 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 (MLS)
Hemocytometers - InCyto Neubauer Improved (DHC-NO1-5)
  
The protocol workflow is as follows: 
A. Isolate lung
B. First layer
C. Second layer
D. Preparing cells for Chromium
  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
    
 
 
 
 
 

[STEPS]
SECTION: Isolate tissue
1. Isolate lung (optional: perfuse lung with DPBS to reduce RBC). Place lung in ice-cold DPBS and transport on ice.
SECTION: Isolate tissue
2. Using sterile forceps, transfer lung tissue to petri dish on ice. Remove excess DPBS using pipet. Mince lung tissue on petri dish on ice for 2 min until fine paste. Vigorously mince tissue using forceps to manipulate the tissue with one hand while using a grinding motion with the razor blade in the other hand.
2 min
SECTION: 1st layer
3. Weigh out 25 mg of tissue on petri dish. Using a sterile razor blade or forceps place 25 mg tissue in 1 mL enzyme mix in 1.5 mL eppendorf tube, incubating on ice.
25 5
SECTION: 1st layer
4. Incubate on ice. Shake tube vigorously every 30 secs. Begin triturating at 2 mins. Triturate 10X every 1.5 minute (first w/tip cut).
30 s90 s
SECTION: 1st layer
5. After 5 min pipet tissue + enzyme mix into new sterile petri dish on ice. Mince 2 min using razor blade to further break up residual chunks of tissue.
5 min2 min
SECTION: 1st layer
6. Pipet digest mix back into 1.5 mL tube. Rinse petri dish with 0.5 mL coll. A/elastase/dispase enzyme mix and pipet into same tube.
0.5 2
SECTION: 1st layer
7. Continue triturating and shaking on ice for 2 additional minutes until you reach 9 minutes total digestion time
2 min
SECTION: 1st layer
8. At 9 min total digest time let tube settle for one min on ice. The chunks of tissue will settle to the bottom of the tube, leaving released cells in the supernatant. Pipet 80% of supernatant onto 30 µM filter on sterile 15 mL conical.
1 min
SECTION: 1st layer
9. Rinse filter w/6 mL ice-cold PBS/BSA 0.04%. Leave filter on 15 mL conical for next steps. 
6 2
SECTION: 2nd layer
10. Add additional 1 mL of 10 mg/mL b. lich enzyme mix to residual clumps of tissue in enzyme in the 1.5 mL tube. 
1 2
SECTION: 2nd layer
11. Continue triturating on ice 10x every 1.5 minute for 10 additional minutes (20 min total time). Shake every 30 sec. 
10 min90 s30 s
SECTION: 2nd layer
12. Pipet entire volume onto same 30 µM filter on 15 mL conical - rinse w/6 mL ice-cold PBS/BSA 0.04%. 
6 2
SECTION: 2nd layer
13. Spin 300 g for five minutes at 4 ºC. Remove all but 100 µL of supernatant - being careful not to disturb pellet.
4 10
SECTION: RBC Lysis
14. Add 900 µL RBC lysis buffer to pellet. Triturate 20X using 1 mL pipet set to 700 µL and incubate for two min on ice.
900 12 min
SECTION: RBC Lysis
15. Add 12 mL ice-cold PBS/BSA 0.04% to 15 mL conical to dilute RBC lysis buffer.
12 2
SECTION: Preparing for Single-Cell Sequencing
16. Spin 15 mL conical 200 g (low-g spin) for 5 min at 4 ºC to pellet cells and leave small debris and platelets in supernatant.
5 min4 10
SECTION: Preparing for Single-Cell Sequencing
17. Remove supernatant and re-suspend in 200 µL ice-cold PBS/BSA 0.04%. 
200 1
SECTION: Preparing for Single-Cell Sequencing
18. Optional: to increase the % of viable cells, at this point in the procedure you can perform dead cell removal using the EasySep dead cell removal kit according to the manufacturer’s instructions.
SECTION: Preparing for Single-Cell Sequencing
19. Examine cells using hemocytometer w/trypan blue. Adjust concentration to 1000 cells / µL for 10X Chromium or 100 cells / µL for DropSeq.