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

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>This protocol is used to dissociate adult (10 week) CD-1 mouse liver "on ice", preserving authentic gene expression profiles. The first layer consists of 3.5 hours rotation at 4 °C in 0.25% trypsin, and the second layer consists of 15 min. in </span><span style = "font-style:italic;">bacillus licheniformis</span><span>, with trituration on ice. The yield is 5000 cells/mg with 97% viability (as measured by trypan blue). </span></div></div>

[STEPS]
?. [Isolate tissue]
Dissect out liver tissue and immerse/transport in ice-cold PBS.
?. [Isolate tissue]
Using sterile forceps, place tissue on petri dish on ice. Remove excess DPBS using pipet. Mince tissue thoroughly on petri dish on ice (~2 min) until fine paste. Manipulate tissue with forceps while mincing with razor blade.
[mince on ice]
?. [First layer of dissociation]
Add 18 mg tissue to 1 mL trypsin enzyme mix. Shake tube vigorously to re-suspend tissue.
[minced tissue]
?. [First layer of dissociation]
Leave digest mix rotating at 4 °C for 3.5 hrs on Fisher tube rotator. Every 45 min (4X total), stop rotation briefly and shake tube vigorously to re-suspend tissue.
[rotate at 4 °C]
[shake vigorously]
?. [First layer of dissociation]
After 3.5 hours, transfer digest mix to 15 mL conical & add 10 mL ice-cold DPBS to dilute trypsin mix. Re-suspend digest mix.
[ice-cold DPBS]
?. [First layer of dissociation]
Spin 300 g for 5 min at 4 °C to pellet cells and tissue chunks. Remove supernatant.
[spin 300 g]
?. [First layer of dissociation]
After tissue chunks have settled to bottom of tube, transfer 75% of supernatant, containing released cells onto 30 µM filter on 50 mL conical. Rinse filter with 6 mL ice-cold PBS/BSA 0.04%. Save filter and flow-through for next steps.
[pipet off supernatant]
[ice-cold PBS/BSA 0.04%]
?. [Second layer of dissociation]
To tissue clumps at bottom of tube, add 1 mL bacillus licheniformis enzyme mix. Triturate 10X.
[bacillus licheniformis enzyme mix]
?. [Second layer of dissociation]
Continue incubating on ice in bacillus licheniformis enzyme mix. Shake every minute and triturate 10X every 2 min for 15 additional minutes (3 hr. 45 min total time) until the majority of clumps are broken up.
[incubate on ice]
[triturate 10x every 2 min]
?. [Second layer of dissociation]
Transfer entire volume of digest mix to the same 30 µM filter. Rinse filter with 5 mL ice-cold PBS/BSA 0.04%.
[ice-cold PBS/BSA 0.04%]
?. [Second layer of dissociation]
Re-suspend and transfer flow-through containing cells to 15 mL conical.
?. [Second layer of dissociation]
Spin 300 g for 5 min at 4 °C. Remove supernatant (down to ~100 µL) and leave in 15 mL conical.
[300 g for 5 min]
?. [RBC Lysis]
Add 1 mL RBC lysis buffer; triturate 20X; let sit 2 min. on ice.
[RBC lysis buffer]
[incubate on ice]
?. [RBC Lysis]
After 2 min., add 5 mL ice-cold PBS/BSA 0.04% and re-suspend cells.
[ice-cold PBS/BSA 0.04%]
?. [Preparing for single cell analysis]
Spin 1800 RPM (low-speed spin) for 3 minutes at 4 °C to pellet cells and leave the majority of debris in supernatant.
[spin 1800 RPM]
?. [Preparing for single cell analysis]
Remove supernatant; re-suspend cells in ~100 µL ice-cold PBS/BSA 0.04% and analyze using hemocytometer with trypan blue. Adjust cell concentration to 1,000 cells/µL for Chromium or 100 cells/µL for DropSeq.
[ice-cold PBS/BSA 0.04%]
?. [First layer of dissociation]
Re-suspend cells and tissue in 1 mL ice-cold DPBS and transfer to 1.5 mL tube. Let chunks settle one minute on ice (released cells should remain in supernatant).
[let chunks settle on ice]
?. [RBC Lysis]
Add re-suspended cells to new 30 µM filter on 50 mL conical; rinse filter with 7 mL ice-cold PBS/BSA 0.04%. Transfer flow-through to 15 mL conical.
[ice-cold PBS/BSA 0.04%]