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

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">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. </div></div>

[STEPS]
?. [Isolate tissue]
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.
[mince tissue on ice]
?. [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.
[minced lung tissue]
?. [1st layer]
Incubate on ice. Shake tube vigorously every 30 secs. Begin triturating at 2 mins. Triturate 10X every 1.5 minute (first w/tip cut).
[shake]
[triturate 10x]
?. [1st layer]
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.
[After 5 min. digesting pipet digest mix into petri dish]
[mince in petri dish]
?. [1st layer]
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.
[rinse petri dish]
?. [1st layer]
Continue triturating and shaking on ice for 2 additional minutes until you reach 9 minutes total digestion time
[triturate on ice]
?. [1st layer]
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.
[settle on ice]
?. [1st layer]
Rinse filter w/6 mL ice-cold PBS/BSA 0.04%. Leave filter on 15 mL conical for next steps.
[ice-cold PBS/BSA 0.04%]
?. [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.
[B. lich enzyme mix]
?. [2nd layer]
Continue triturating on ice 10x every 1.5 minute for 10 additional minutes (20 min total time). Shake every 30 sec.
[digest on ice]
[triturate 10x]
[shake]
?. [2nd layer]
Spin 300 g for five minutes at 4 ºC. Remove all but 100 µL of supernatant - being careful not to disturb pellet.
[spin at 300 g]
?. [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.
[RBC lysis buffer]
[incubate on ice]
?. [RBC Lysis]
Add 12 mL ice-cold PBS/BSA 0.04% to 15 mL conical to dilute RBC lysis buffer.
[ice-cold PBS/BSA 0.04%]
?. [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.
[spin at 200 g]
?. [Preparing for Single-Cell Sequencing]
Remove supernatant and re-suspend in 200 µL ice-cold PBS/BSA 0.04%.
[ice-cold PBS-BSA 0.04%]
?. [2nd layer]
Pipet entire volume onto same 30 µM filter on 15 mL conical - rinse w/6 mL ice-cold PBS/BSA 0.04%.
[ice-cold PBS/BSA 0.04%]
?. [Preparing for Single-Cell Sequencing]
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.
?. [Preparing for Single-Cell Sequencing]
Examine cells using hemocytometer w/trypan blue. Adjust concentration to 1000 cells / µL for 10X Chromium or 100 cells / µL for DropSeq.
?. [Isolate tissue]
Isolate lung (optional: perfuse lung with DPBS to reduce RBC). Place lung in ice-cold DPBS and transport on ice.