Adult mouse lung cell dissociation (on ice)
Andrew Potter
Abstract
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.
Steps
Isolate tissue
Isolate lung (optional: perfuse lung with DPBS to reduce RBC). Place lung in ice-cold DPBS and transport on ice.
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.
0h 2m 0s
1st layer
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
Incubate on ice. Shake tube vigorously every 30 secs. Begin triturating at 2 mins. Triturate 10X every 1.5 minute (first w/tip cut).
0h 0m 30s``0h 1m 30s
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.
0h 5m 0s``0h 2m 0s
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
Continue triturating and shaking on ice for 2 additional minutes until you reach 9 minutes total digestion time
0h 2m 0s
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.
0h 1m 0s
Rinse filter w/6 mL ice-cold PBS/BSA 0.04%. Leave filter on 15 mL conical for next steps.
6
2nd layer
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
Continue triturating on ice 10x every 1.5 minute for 10 additional minutes (20 min total time). Shake every 30 sec.
0h 10m 0s``0h 1m 30s``0h 0m 30s
Pipet entire volume onto same 30 µM filter on 15 mL conical - rinse w/6 mL ice-cold PBS/BSA 0.04%.
6
Spin 300 g for five minutes at 4 ºC. Remove all but 100 µL of supernatant - being careful not to disturb pellet.
4
RBC Lysis
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``0h 2m 0s
Add 12 mL ice-cold PBS/BSA 0.04% to 15 mL conical to dilute RBC lysis buffer.
12
Preparing for Single-Cell Sequencing
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.
0h 5m 0s``4
Remove supernatant and re-suspend in 200 µL ice-cold PBS/BSA 0.04%.
200
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.
Examine cells using hemocytometer w/trypan blue. Adjust concentration to 1000 cells / µL for 10X Chromium or 100 cells / µL for DropSeq.
