Oct 22, 2021
Version 3
P1 Kidney Cold-Active Protease Single Cell Dissociation V.3
- Andrew Potter1,
- Steve Potter1
- 1CCHMC
Protocol Citation: Andrew Potter, Steve Potter 2021. P1 Kidney Cold-Active Protease Single Cell Dissociation. protocols.io https://dx.doi.org/10.17504/protocols.io.bzeap3aeVersion created by Andrew Potter
Manuscript citation:
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it’s working
Created: October 22, 2021
Last Modified: October 22, 2021
Protocol Integer ID: 54434
Keywords: CAP, cold-active protease, bacillus licheniformis, single cell dissociation, kidney
Abstract
Method used to derive single cell suspension from P1 mouse kidneys on ice, generating a cell suspension with greatly reduced artifact gene expresion changes and suitable for downstream analysis using 10x Chromium or DropSeq.
Guidelines
Storage Conditions of Reagents
| A | B | |
| Reagent | Storage Condition | |
| DPBS (Thermofisher, 14190144) | 4°C | |
| 1 M CaCl2 | room temp. | |
| BSA (Sigma, A8806) | 4°C | |
| Protease from Bacillus Licheniformis (Sigma, P5380) | Store 100 μL aliquots (100 mg/mL) in DPBS at -80°C | |
| DNAse 1 (StemCell, 07469) | Store 10 μL aliquots (250 U/10 μL) in DPBS at -80°C |
Required Equipment
| Equipment | Supplier | Catalog no. | |
| gentleMACS dissociator | Miltenyi | 130-093-235 |
The protocol workflow is as follows:
A. Isolate Kidney
B. Initial digestion: triturate on ice
C. Perform gentleMACS
D. Continue triturating on ice
F. Preparing cells for Chromium/DropSeq
BEFORE STARTING
Prepare Bacillus Licheniformis enzyme mix just prior to starting dissociation:
| A | B | C | |
| Volume (µl) | Reagent | Final concentration | |
| 890 | DPBS | 1X | |
| 5 | 1 M CaCl2 | 5 mM | |
| 5 | DNAse 1 (250 U/10 μL) | 125 U / mL | |
| 100 | B. Lich (100 mg/mL) | 10 mg/mL |
+25 mg tissue / 1 mL enzyme mix
To prepare 0.01% BSA/PBS:
Make stock of 10% BSA in DPBS and store at -20 °C. To make PBS/BSA 0.01% aliquot 50 mL of DPBS in 50 mL conical and pipet in 50 µL of 10% BSA stock.
Prepare 10% FBS/PBS with heat-inactivated FBS.
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 mg
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.
00:07:00
00:02:00
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 °C
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 °C
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.
00:08:00
00:02:00
Add 3 mL ice-cold 10% FBS/PBS to digest mix in C-tube to inhibit the protease.
3 mL ice-cold 10% FBS/PBS
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 °C
00:05:00 300 g spin
2 mL re-suspend in PBS/BSA
5m
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 mL rinse filter with PBS/BSA
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 °C
00:05:00 300 g spin
10 mL PBS/BSA
5m
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.