Adult mouse kidney dissociation (on ice)
Andrew Potter
Abstract
Protocol for adult (8-10 week) mouse kidney dissociation performed on ice to reduce artifact gene expression. The protocol is based on our protocol published in Development for P1 mouse kidney , however this protocol includes two layers to provide additional enzymatic digestion. Cell viability is ~80% with major kidney cell types represented.
Before start
-Prepare enzyme mixes and leave on ice.
-Cool centrifuges to 4 °C.
-Isolate and transport tissue in ice-cold DPBS.
Attachments
Steps
Isolate kidney
Perfuse kidneys to remove RBC. Extract and isolate kidneys in ice-cold PBS. Leave kidneys in ice-cold PBS until ready to dissociate (Up to 1 hr).
Coarsely mince tissue in PBS.
0h 2m 0s
Layer 1
Weigh out 25 mg coarsely minced tissue for each set of kidneys (remove PBS before weighing).
25
Continue mincing kidneys on top of petri dish, on ice, using razor blade in small vol. (~50 µL) PBS. (1-2 min) until fine paste.
50µL
Prepare a separate 1 mL aliquot of B. Lich enzyme mix for each set of adult kidneys (prepare on ice). Use p200 w/cut tip to transfer minced kidney tissue from petri dish to tube of enzyme mix.
1mL
Incubate digest mix for 10 min on ice with trituration and shaking. Triturate 15 strokes using 1 mL pipet set to 600 µL every 2 min; shake vigorously every min.
0h 1m 0s
0h 2m 0s
0h 10m 0s
After 10 min, let tissue chunks settle on ice for 1 min. Save supernatant (700 µL, 70%) and apply to 30 µM filter on 15 mL conical. Rinse filter w/5 mL 10% FBS/PBS. Leave filter on 15 mL conical for the next layer.
0h 1m 0s
700µL
5mL
Layer 2
Add additional 700 µL B. Lich enzyme mix to residual tissue chunks. Continue incubating on ice 15-20 min with shaking and trituration, until tubules and glomeruli are fully broken up.
700µL
0h 1m 0s
0h 2m 0s
0h 20m 0s
Once digestion is adequate (tubules / glomeruli are broken up), triturate and add entire digest mix to same 30 µM filter as used in previous step on 15 mL conical.
Spin 15 mL conical with combined flow-through from layer 1 and layer 2 (isolated cells) 300 g for 5 min at 4 °C.
0h 5m 0s
Prepare cells for 10X Chromium / scRNA-Seq
Discard supernatant. If necessary, perform RBC removal according to manufacturers instructions.
Re-suspend cells in 10 mL 10% FBS/PBS. Spin 300 g for 5 min at 4 °C.
10mL
0h 5m 0s
Discard supernatant. Re-suspend cells in 500 µL 10% FBS/PBS (or other compatible buffer). Analyze cell viability and concentration using trypan blue dye exclusion with a hemocytometer. Adjust cell concentration to 700-1,200 cells/µL for 10X single cell 3'v3.1.
500µL
