Adult mouse pancreas cell dissociation (on ice)
Andrew Potter
Abstract
Note: In testing several different enzymes to dissociate pancreas tissue on ice, Collagenase Type 4 appeared to be most effective. However, the results of the dissociation have been variable, with significant debris in some preps, and cell type representation has not validated using 10X or flow; further optimization may be needed with this protocol.
This procedure is used to dissociate adult (10 wk.) mouse pancreas into single cells. The procedure is carried out on ice in order to maintain a more authentic gene expression profile. It is a two-layered dissociation, with each layer consisting of 5 mg/mL type 4 collagenase. The yield is 4400 cells/mg with 94% viability.
Before start
-Prepare enzyme mixes and leave on ice.
-Cool centrifuges to 4 °C.
-Isolate and transport tissue in ice-cold DPBS.
Attachments
Steps
Dissect pancreas and place in ice-cold PBS.
Mince tissue thoroughly on petri dish on ice (2 min) until fine paste.
0h 2m 0s
Weigh out 18 mg tissue and add to tube with 1 mL Type 4 collagenase enzyme mix.
18
Incubate on ice. Shake vigorously every 30 seconds for the first two min to re-suspend tissue.
0h 0m 30s
At two mins, begin triturating 10x every min. Continue triturating on ice for 20 min.
0h 20m 0s
After incubating 20 min, let chunks settle for 1 min on ice.
0h 1m 0s
Pipet top 75% (750 µL) of supernatant containing released cells onto a 30 µM filter on a 50 mL conical, on ice.
750
Rinse filter with 5 mL ice-cold PBS/BSA 0.04%. Save filter and flow-through for next steps.
5
To residual tissue chunks add additional 1 mL type 4 collagenase enzyme mix.
1
Continue triturating on ice 10x every min for 30 additional min (50 min total digest time).
0h 30m 0s
Triturate and add entire volume to same 30 µM filter on 50 mL conical. Rinse filter w/5 mL ice-cold PBS/BSA 0.04%.
5
Transfer flow-through to 15 mL conical. Spin 300 g for 5 min at 4 °C.
0h 5m 0s
Remove supernatant and re-suspend in 100 µL ice-cold PBS/BSA 0.04%.
Add 900 µL RBC lysis buffer. Triturate 20x and incubate 2 min. on ice.
0h 2m 0s
Add 10 mL ice-cold PBS/BSA 0.04% to dilute RBC lysis buffer.
Spin 300 g for 5 min at 4 °C. Remove supernatant.
0h 5m 0s
Re-suspend in 100 µL ice-cold PBS/BSA 0.04%. Analyze viability and yield using a hemocytometer with trypan blue. Adjust concentration to 1,000 cells/µL for Chromium or 1,000 cells/µL for DropSeq.
100
