Feb 27, 2019
Version 3
Adult mouse lung cell dissociation (on ice) V.3
- 1CCHMC
- Human Cell Atlas Method Development Community
Protocol Citation: Andrew Potter 2019. Adult mouse lung cell dissociation (on ice). protocols.io https://dx.doi.org/10.17504/protocols.io.ymgfu3w
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: February 26, 2019
Last Modified: February 27, 2019
Protocol Integer ID: 20872
Keywords: lung, dissociation, single cell, CAP
Abstract
This protocol was used to dissociate adult (8-10 wk) mouse lung tissue. The entire procedure is carried out on ice (to reduce artifact gene expression changes) and takes about half an hour. The yield was 16,240 non-RBC/mg tissue with 87% viability.
Guidelines
Enzyme Mixes
Coll. A/Elastase/Dispase Enzyme Mix (1.5 mL --> save 0.5 mL of coll./elastase/dispase mix in separate 1.5 mL tube. )
90 µL Collagenase A 100 mg/mL – 6 mg/mL final (Sigma, 10103578001)
150 µL elastase 43 u/mL - 4.3 u/mL final (Worthington, LS002292)
150 µL Dispase 90 u/mL – 9 u/mL final (Worthington, LS02100)
7.5 µL 1 M CaCl2 – 5 mM final
7.5 µL DNAse (125 U/mL) (Applichem, A3778)
1095 µL PBS
B. Lich enzyme mix (1 mL)
899 µL DPBS (no Ca, no Mg) (Thermo Fisher, 14190144)
1 µL 0.5 M EDTA - 0.5 mM final conc. (Sigma, A8806)
100 µL Bacillus Licheniformis 100 mg/mL - 10 mg/mL final conc. (Sigma, P5380)
Preparing stock aliquots of reagents:
Enzyme stocks are made up in DPBS. I make up 100 mg/mL stock of Collagenase A and Bacillus Licheniformis. For elastase, I prepare 43 u/mL stock, and for dispase a 90 u/mL stock. Enzymes mixes are aliquoted in 150 µL (elastase, depase) or 100 µL (Collagenase A, Bacillus Licheniformis) and stored at -80 ºC. For the CaCl2, I prepare a 1 M stock and autoclave. The DNAse is diluted in DPBS, aliquoted and stored at -80 ºC.
Required reagents:
Red Blood Cell Lysis Buffer (Sigma, R7757) - store at RT
Trypan blue solution, 0.4% (ThermoFisher, 15250061)
(Optional) Dead Cell Removal Kit:
EasySep dead cell removal (Annexin V) kit (cat. #17899)
EasySep Magnet (cat. #18000)
Required Equipment & Consumables:
Centrifuges for 1.5 mL and 15 mL conicals (MLS)
Pipettes and pipet tips (MLS)
15, 50 ml Conicals (MLS)
1.5 mL tubes (MLS)
30 µM filters - Miltenyi (130-098-458)
Petri dishes (MLS)
Razor blades (MLS)
Ice bucket w/ice (MLS)
Hemocytometers - InCyto Neubauer Improved (DHC-NO1-5)
The protocol workflow is as follows:
A. Isolate lung
B. First layer
C. Second layer
D. Preparing cells for Chromium
Materials
MATERIALS
RBC Lysis Buffer Merck MilliporeSigma (Sigma-Aldrich)Catalog #R7757
DNAse AppliChemCatalog #A3778
BSA
Before start
-Prepare enzyme mixes and leave on ice.
-Cool centrifuges to 4 °C.
Isolate tissue
Isolate tissue
Isolate lung (optional: perfuse lung with DPBS to reduce RBC). Place lung in ice-cold DPBS and transport on ice.
Using sterile forceps, transfer lung tissue to petri dish on ice. Remove excess DPBS using pipet. Mince lung tissue on petri dish on ice for 2 min until fine paste. Vigorously mince tissue using forceps to manipulate the tissue with one hand while using a grinding motion with the razor blade in the other hand.
00:02:00 mince tissue on ice
1st layer
1st layer
Weigh out 25 mg of tissue on petri dish. Using a sterile razor blade or forceps place 25 mg tissue in 1 mL enzyme mix in 1.5 mL eppendorf tube, incubating on ice.
25 mg minced lung tissue
Incubate on ice. Shake tube vigorously every 30 secs. Begin triturating at 2 mins. Triturate 10X every 1.5 minute (first w/tip cut).
00:00:30 shake 00:01:30 triturate 10x
After 5 min pipet tissue + enzyme mix into new sterile petri dish on ice. Mince 2 min using razor blade to further break up residual chunks of tissue.
00:05:00 After 5 min. digesting pipet digest mix into petri dish 00:02:00 mince in petri dish
Pipet digest mix back into 1.5 mL tube. Rinse petri dish with 0.5 mL coll. A/elastase/dispase enzyme mix and pipet into same tube.
0.5 mL rinse petri dish
Continue triturating and shaking on ice for 2 additional minutes until you reach 9 minutes total digestion time
00:02:00 triturate on ice
At 9 min total digest time let tube settle for one min on ice. The chunks of tissue will settle to the bottom of the tube, leaving released cells in the supernatant. Pipet 80% of supernatant onto 30 µM filter on sterile 15 mL conical.
00:01:00 settle on ice
Rinse filter w/6 mL ice-cold PBS/BSA 0.04%. Leave filter on 15 mL conical for next steps.
6 mL ice-cold PBS/BSA 0.04%
2nd layer
2nd layer
Add additional 1 mL of 10 mg/mL b. lich enzyme mix to residual clumps of tissue in enzyme in the 1.5 mL tube.
1 mL B. lich enzyme mix
Continue triturating on ice 10x every 1.5 minute for 10 additional minutes (20 min total time). Shake every 30 sec.
00:10:00 digest on ice 00:01:30 triturate 10x 00:00:30 shake
Pipet entire volume onto same 30 µM filter on 15 mL conical - rinse w/6 mL ice-cold PBS/BSA 0.04%.
6 mL ice-cold PBS/BSA 0.04%
Spin 300 g for five minutes at 4 ºC. Remove all but 100 µL of supernatant - being careful not to disturb pellet.
4 °C spin at 300 g
RBC Lysis
RBC Lysis
Add 900 µL RBC lysis buffer to pellet. Triturate 20X using 1 mL pipet set to 700 µL and incubate for two min on ice.
900 µL RBC lysis buffer 00:02:00 incubate on ice
Add 12 mL ice-cold PBS/BSA 0.04% to 15 mL conical to dilute RBC lysis buffer.
12 mL ice-cold PBS/BSA 0.04%
Preparing for Single-Cell Sequencing
Preparing for Single-Cell Sequencing
Spin 15 mL conical 200 g (low-g spin) for 5 min at 4 ºC to pellet cells and leave small debris and platelets in supernatant.
00:05:00 spin at 200 g 4 °C
Remove supernatant and re-suspend in 200 µL ice-cold PBS/BSA 0.04%.
200 µL ice-cold PBS-BSA 0.04%
Optional: to increase the % of viable cells, at this point in the procedure you can perform dead cell removal using the EasySep dead cell removal kit according to the manufacturer’s instructions.
Examine cells using hemocytometer w/trypan blue. Adjust concentration to 1000 cells / µL for 10X Chromium or 100 cells / µL for DropSeq.