Nov 16, 2023

Public workspaceCell isolation from dorsal mouse skin for single-cell RNA-seq

DOI
dx.doi.org/10.17504/protocols.io.yxmvmn8m9g3p/v1
  • Cenk Celik1,
  • Stella Yue Ting Lee1,
  • Guillaume Thibault1
  • 1Nanyang Technological University
Cenk Celik
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DOI: dx.doi.org/10.17504/protocols.io.yxmvmn8m9g3p/v1
Protocol Citation: Cenk Celik, Stella Yue Ting Lee, Guillaume Thibault 2023. Cell isolation from dorsal mouse skin for single-cell RNA-seq. protocols.io https://dx.doi.org/10.17504/protocols.io.yxmvmn8m9g3p/v1
Manuscript citation:
Cenk Celik, Stella Tue Ting Lee, Frederick Reinhart Tanoto, Mark Veleba, Kimberly Kline, Guillaume Thibault (2024) Decoding the complexity of delayed wound healing following Enterococcus faecalis infection eLife 13:RP95113

https://doi.org/10.7554/eLife.95113.3
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: August 07, 2022
Last Modified: November 16, 2023
Protocol Integer ID: 68336
Funders Acknowledgements:
NMRC
Grant ID: MOH-000566​
Abstract
We identified heterogeneity of the skin cell populations upon Enterococcus faecalis infection.
Materials
Consumables
  • Hank's Balanced Salt Solution Ca2+/Mg2+-free
  • Dulbecco's Phosphate Buffered Saline Ca2+/Mg2+-free
  • Dispase
  • Collagenase type I
  • Liberase TM Research Grade
  • 0.25% Trypsin/EDTA
  • Bovine Serum Albumin
  • Trypan Blue
  • Ice
Instruments
  • CO2 supplemented 37°C incubator
  • Chromium Instrument/X, 10X Genomics
  • Countess 3 Automated Cell Counter, Invitrogen
  • 15ml and 50ml tubes-compatible centrifuge
  • 1.5ml tube-compatible centrifuge
Surgical tools
  • 6-mm punch
  • Scissors
  • Forceps
  • Bistouri
  • Blades G23
  • 10mL syringes
  • 21G needles
Other labware
  • 50ml centrifuge tubes
  • 15ml centrifuge tubes
  • 1.5ml Protein LoBind tubes
  • Disposable haemacytometer (4 per sample)
  • 10mm dishes
  • 6-well plates
  • Pasteur pipettes
Protocol materials
ReagentDispase II, powderThermo FisherCatalog #17105041
Reagent1X Dulbecco’s Phosphate Buffered Saline (DPBS) Thermo Fisher ScientificCatalog #14190094
ReagentCollagenase Type I powderThermo Fisher ScientificCatalog #17100017
ReagentHanks Balanced Salt Solution (HBSS) without Ca2 Mg2 Thermo Fisher ScientificCatalog #88284
ReagentHanks Balanced Salt Solution (HBSS) without Ca2 Mg2 Thermo Fisher ScientificCatalog #88284
ReagentLiberase TMMerck MilliporeSigma (Sigma-Aldrich)Catalog #000000005401119001
ReagentHanks Balanced Salt Solution (HBSS) without Ca2 Mg2 Thermo Fisher ScientificCatalog #88284
ReagentBovine Serum AlbuminMerck MilliporeSigma (Sigma-Aldrich)Catalog #A7030
ReagentHanks Balanced Salt Solution (HBSS) without Ca2 Mg2 Thermo Fisher ScientificCatalog #88284
Reagent1X Dulbecco’s Phosphate Buffered Saline (DPBS) Thermo Fisher ScientificCatalog #14190094
ReagentHanks Balanced Salt Solution (HBSS) without Ca2 Mg2 Thermo Fisher ScientificCatalog #88284
ReagentTrypsin EDTAGibco - Thermo Fisher ScientificCatalog #25-051-CI.
ReagentHanks Balanced Salt Solution (HBSS) without Ca2 Mg2 Thermo Fisher ScientificCatalog #88284
ReagentStandard Biopsy Punches, Disposable Standard biopsy punch; 6mmThermo FisherCatalog #12460412
Reagentscalpel blades
Reagent10 mL syringesBecton Dickinson (BD)Catalog #BD 309695
ReagentFalcon® 70 µm cell strainerCorningCatalog #352350
Reagent10 mL syringesBecton Dickinson (BD)Catalog #BD 309695
ReagentFalcon® 70 µm cell strainerCorningCatalog #352350
ReagentFalcon® 40 µm Cell StrainerCorningCatalog #352340
Enzyme solutions
Enzyme solutions
20m
20m
Dispase Solution

Dissolve 0.5g ofReagentDispase II, powderBecton Dickinson (BD)Catalog #17105041 in a 50ml ofReagent1X Dulbecco’s Phosphate Buffered Saline (DPBS) Becton Dickinson (BD)Catalog #14190094 to make Concentration10 mg/mL of Dispase II solution

2m
Filter the solution by using a 0.2µm filter
2m
Aliquot 1.25mL in tubes and store at Temperature-20 °C
5m
Collagenase type I Solution

Dissolve 1g ofReagentCollagenase Type I powderBecton Dickinson (BD)Catalog #17100017 in 4mL of ReagentHanks Balanced Salt Solution (HBSS) without Ca2 Mg2 Becton Dickinson (BD)Catalog #88284 to make a Concentration250 mg/mL collagenase type I solution

2m
Make a stock solution

i.e. 67500U/mL x 4mL = 50U/µL x Vmax
Vmax = 5.4mL
5.4 - 4.0 = 1.4mL to add the stock to make the stock solution
2m
Filter the solution by using a 0.2µm filter
2m
Aliquot 20µL in tubes and store at Temperature-20 °C

5m
Liberase TM Solution

Punch through the cap with a syringe to add 2mL of ReagentHanks Balanced Salt Solution (HBSS) without Ca2 Mg2 Becton Dickinson (BD)Catalog #88284 into 5mg ReagentLiberase TMBecton Dickinson (BD)Catalog #000000005401119001 bottle to make Concentration2.5 mg/mL liberase solution

2m
Dilute into Concentration0.2 mg/mL in ReagentHanks Balanced Salt Solution (HBSS) without Ca2 Mg2 Becton Dickinson (BD)Catalog #88284 (1.04 Wünsch unit/mL)

2m
Filter the solution by using a 0.2µm filter
2m
Aliquot 750µL in tubes and store at Temperature-20 °C

5m
Working Solutions
Working Solutions
2m
2m
Working Solution 1 (WS1)

Make a Concentration0.5 Mass / % volume ofReagentBovine Serum AlbuminBecton Dickinson (BD)Catalog #A7030 in 50mL of ReagentHanks Balanced Salt Solution (HBSS) without Ca2 Mg2 Becton Dickinson (BD)Catalog #88284
2m
Filter the solution by using a 0.2µm filter and keep it at Temperature4 °C

2m
Working Solution 2 (WS2)

Make a Concentration0.04 Mass / % volume of Bovine serum albumin solution in 50mL of Reagent1X Dulbecco’s Phosphate Buffered Saline (DPBS) Becton Dickinson (BD)Catalog #14190094

2m
Filter the solution by using a 0.2µm filter and keep it at Temperature4 °C
2m
Enzyme Cocktail 1 (EC1)–4 samples

From the enzyme stocks that have been prepared before, mix:
  • 1.25mL of Dispase
  • 0.75mL of Liberase
  • 20µL of type I Collagenase
in a 15mL tube
3m
Add 8.0 mL of
ReagentHanks Balanced Salt Solution (HBSS) without Ca2 Mg2 Becton Dickinson (BD)Catalog #88284

1m
Warm up in a Temperature37 °C water bath before using
30m
Enzyme Cocktail 2 (EC2)–4 samples

Dilute theReagentTrypsin EDTABecton Dickinson (BD)Catalog #25-051-CI. in ReagentHanks Balanced Salt Solution (HBSS) without Ca2 Mg2 Becton Dickinson (BD)Catalog #88284 to make a Concentration0.05 Mass / % volume of trypsin/EDTA solution

2m
Warm up in a Temperature37 °C water bath before using
30m
Tissue Dissociation and Cell Isolation Procedure
Tissue Dissociation and Cell Isolation Procedure
7m
7m
Punch out the skins from mice using a sterileReagentStandard Biopsy Punches, Disposable Standard biopsy punch; 6mmBecton Dickinson (BD)Catalog #12460412 and immediately float on WS1 on ice

5m
Peel off any fat tissue beneath the skin using a bladeReagentscalpel bladesBecton Dickinson (BD)
2m
Mince the fat tissue-cleaned skin samples into smaller pieces using sterile scissors while holding the tissue with sterile forceps on ice
2m
Incubate the minced tissues in 2 ml of EC1 in a 6-well plate at a Temperature37 °C incubator with a 5% CO2 supplementation for two hours

Duration02:00:00
2h
Incubation
Digestion
Shake the plate orbitally in the incubator every Duration00:15:00 for better digestion

15m
Critical
Remove a piston from a sterile,Reagent10 mL syringesBecton Dickinson (BD)Catalog #BD 309695 to smash the partly-digested tissues in the plate
1m
Place aReagentFalcon® 70 µm cell strainerBecton Dickinson (BD)Catalog #352350 into a 50mL tube and sift through the cell/tissue mixture

1m
Wash the EC1-digested tissue on the cell strainer with WS1 thrice
3m
Wash
Save the flow-through on ice
Invert the cell strainer with tissue remnants and place it on a sterile 6-well plate
1m
From the sieve-through side of the cell strainer, add 2mL of EC2 aiming at tissue remnants
1m
Incubate the undigested tissue at a Temperature37 °C incubator with a 5% CO2 supplementation for Duration00:15:00
15m
Incubation
Digestion
Remove a piston from a sterile,Reagent10 mL syringesBecton Dickinson (BD)Catalog #BD 309695 to smash the partly-digested tissues in the plate
1m
Place a sterileReagentFalcon® 70 µm cell strainerBecton Dickinson (BD)Catalog #352350 into the 50mL tube obtained at Step 15 to pool and sift through the cell/tissue mixture
1m
Mix
Wash the trypsin-digested tissue on the cell strainer with WS1 thrice
1m
Wash
Place a sterileReagentFalcon® 40 µm Cell StrainerBecton Dickinson (BD)Catalog #352340 into a clean 50mL tube and sift through the cell suspension

1m
Centrifuge the pooled cell suspensions at Centrifigation300 x g, 20°C, 00:10:00
10m
Carefully aspirate the supernatant and resuspend the pellet in 1mL of WS2
2m
Pipetting
Centrifuge the pooled cell suspensions again at Centrifigation300 x g, 20°C, 00:05:00
5m
Carefully aspirate the supernatant and resuspend the pellet in 500µL of WS2
1m
Pipetting
Determine the cell viability by mixing 10µl of the cell suspensions with 10µl of trypan blue
Equipment
Countess 3 FL Automated Cell Counter
NAME
Automated Cell Counter
TYPE
Thermofisher scientific
BRAND
AMQAF2000
SKU
https://www.thermofisher.com/th/en/home/life-science/cell-analysis/cell-analysis-instruments/automated-cell-counters/models/countess-3-fl.html
LINK

2m
Analyze
Take at least four counts per sample
8m
Critical
Remember to tick "Trypan Blue correction" on the Countess cell counter
1m
Critical
Average cell numbers for each sample and dilute each cell suspension with WS2 to adjust the cell number to 700-1200 cells/µL as indicated in 10X Genomics Chromium Next GEM Single Cell 3ʹ Reagent Kits v3.1 Protocol (CG000315)
5m
Aim for cell viability above 70% for each sample
Expected result
Cell viability: 70% per count

Critical
If the cell viability is below 70%, you may choose to remove dead cells using a dead cell removal kit
Optional