P1 Kidney Cold-Active Protease Single Cell Dissociation

Extract & isolate P1 kidneys in ice-cold PBS.

Mince kidneys on top of petri dish, on ice, using razor blade.

Weigh out 25 mg of tissue for each tube of B. Lich. enzyme mix (2 tubes total).

25

Incubate tissue + enzyme on ice for 7 minutes while triturating 15 strokes using 1 mL pipet every 2 minutes set to 700 µL - first with tip cut off.

0h 7m 0s

0h 2m 0s

After 7 minutes, take the digest mix (combine the two tubes) and pipet into Miltenyi C-tube (placed on ice); take C-tube to gentleMACS placed in 4° cold room. Run program brain_03 two times.

4

After MACS, briefly quick spin the MACS tube (to 300 G) at 4 °C to ensure contents are in the bottom of the tube. 

4

Re-suspend and visualize cells using scope by taking small aliquot and using a slide; continue digesting cells in C-tube on ice for 8 additional minutes while triturating every 2 min 15 strokes using a 1 mL pipet.

0h 8m 0s

0h 2m 0s

Add 3 mL ice-cold 10% FBS/PBS to digest mix in C-tube to inhibit the protease. 

3

Transfer digest mix to a 15 mL conical. Spin 300 G for 5 minutes at 4 °C; discard supernatant; re-suspend cell pellet in 2 mL ice-cold PBS/BSA.

4

0h 5m 0s

2

Filter re-suspended cells using 30 uM filter on sterile 15 mL conical on ice - rinse filter with 8 mL ice-cold PBS/BSA.

8

Spin 15 mL conical tube containing filtered cells 300 G for 5 minutes at 4 °C; discard supernatant and re-suspend pellet in 10 mL ice-cold PBS/BSA.

4

0h 5m 0s

10

Repeat rinse/spin in ice-cold PBS/BSA.

Remove supernatant and re-suspend in 1-2 mL ice-cold PBS/BSA.

Examine using hemocytometer and adjust concentration to 100 cells/uL for DropSeq or 1,000 cells/µL for 10X Chromium.