Feb 13, 2019

Public workspaceCell dissociation from airway biopsies with cold-active protease for single-cell RNA-seq

DOI
dx.doi.org/10.17504/protocols.io.x3efqje
  • 1Université Côte d'Azur, CNRS, IPMC, 06560 Valbonne, France
  • Human Cell Atlas Method Development Community
Laure-Emmanuelle Zaragosi
Icon indicating open access to content
QR code linking to this content
DOI: dx.doi.org/10.17504/protocols.io.x3efqje
Protocol Citation: Laure-Emmanuelle Zaragosi, Pascal Barbry 2019. Cell dissociation from airway biopsies with cold-active protease for single-cell RNA-seq. protocols.io https://dx.doi.org/10.17504/protocols.io.x3efqje
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 12, 2019
Last Modified: February 13, 2019
Protocol Integer ID: 20294
Keywords: bronchial biopsy, airway epithelium, single-cell, dissociation, cold-active protease
Abstract
This protocol provides details on the cell dissociation that should be performed to obtain single-cell suspensions from airway biopsies.
Biopsies may come from tracheal, bronchial or nasal epithelium.
Cell dissociation is performed at 4°C to avoid gene expression alterations and maximize viability. 
The typical cell number recovery is 40 000 cells for one biopsy.
Cell suspensions are suitable for single-cell RNA-sequencing protocols.
Guidelines
Storage Conditions of Reagents 
Reagent Storage Condition 
HBSS4°C
20 mM EDTA room temperature
BSA (Sigma, A8806)4°C
Protease from Bacillus Licheniformis (Sigma, P5380) Store 100 μL aliquots (100 mg/mL) in DPBS at -80°C
Hoechst 33342 (10 mg/mL) 4°C
NucGreen™ Dead 488 ReadyProbes™ Reagent room temperature
Required Equipment
Equipment Supplier Catalog no.
Countess II FL automated cell counter Thermo Fisher Scientific AMQAF1000
The protocol workflow is as follows:  1. Perform airway biopsies in the desired zone 2. Dissociation: mince with scalpel then triturate on ice in dissociation buffer 3. Remove red blood cells if necessary 4. Prepare cells for Chromium/DropSeq All steps should be performed on ice or at 4°C Use wide-bore 1 mL pipet tip for all biopsy transfers.
Materials
MATERIALS
ReagentEDTA
Reagent23G NeedlesCatalog #4657667
ReagentProtease from Bacillus Licheniformis SigmaCatalog #P5380
ReagentQuick-Read 10 Chamber SlideGlobe ScientificCatalog #3805
ReagentCountess™ Cell Counting Chamber SlidesCatalog #C10314
ReagentDPBS no calcium, no magnesiumInvitrogen - Thermo FisherCatalog #14190136
Reagent21G needleVWR international LtdCatalog #BD-305165
ReagentHBSSGibco - Thermo FischerCatalog #14060040
STEP MATERIALS
ReagentQuick-Read 10 Chamber SlideGlobe ScientificCatalog #3805
ReagentAmmonium Chloride Solution 100 mL Stemcell TechnologiesCatalog #7800
ReagentFlowmi cell strainerCatalog #H13680-0040
ReagentHoechst 33342, Trihydrochloride, Trihydrate - 10 mg/mL Solution in WaterThermo Fisher ScientificCatalog #H3570
ReagentNucGreen™ Dead 488 ReadyProbes™ ReagentThermo Fisher ScientificCatalog #R37109
Protocol materials
ReagentCountess™ Cell Counting Chamber SlidesCatalog #C10314
Materials
Reagent21G needleVWR International (Avantor)Catalog #BD-305165
Materials
ReagentProtease from Bacillus Licheniformis Merck MilliporeSigma (Sigma-Aldrich)Catalog #P5380
Materials
ReagentDPBS no calcium, no magnesiumInvitrogen - Thermo FisherCatalog #14190136
Materials
ReagentQuick-Read 10 Chamber SlideGlobe ScientificCatalog #3805
Materials
ReagentQuick-Read 10 Chamber SlideGlobe ScientificCatalog #3805
Materials
ReagentEDTA
Materials
ReagentNucGreen™ Dead 488 ReadyProbes™ ReagentThermo Fisher ScientificCatalog #R37109
Materials, Step 25
ReagentAmmonium Chloride Solution 100 mL STEMCELL Technologies Inc.Catalog #7800
Materials, Step 11
Reagent23G NeedlesCatalog #4657667
Materials
ReagentFlowmi cell strainerCatalog #H13680-0040
Materials, Step 21
ReagentHoechst 33342, Trihydrochloride, Trihydrate - 10 mg/mL Solution in WaterThermo Fisher ScientificCatalog #H3570
Materials, Step 25
ReagentHBSSGibco - Thermo Fisher ScientificCatalog #14060040
Materials
Safety warnings
Samples coming from patients with undetermined viral status should be process in cell culture rooms with the appropriate safety level.
Before start
Prepare Bacillus Licheniformis enzyme mix just prior to starting dissociation:
Volume (µl)Reagent Final concentration 
 850DPBS 1X
 50 20 mM EDTA 0.5 mM
 100 Protease from B. Licheniformis (100 mg/mL) 10 mg/mL
Prepare Inactivation buffer: 
Make stock of 10% BSA in HBSS and store at -20 °C.
To make HBSS/BSA 2% aliquot 40 mL of HBSS in 50 mL conical and pipet in 10 mL of 10% BSA stock.
Prepare Wash buffer: 
To make HBSS/BSA 1% aliquot 20 mL of HBSS in 50 mL conical and pipet in 20 mL of HBSS/BSA 2%.

Prepare Resuspension buffer: 
To make HBSS/BSA 0.05% aliquot 1 mL of HBSS/BSA 2% in 50 mL conical and pipet in 39 mL of HBSS.
Prepare cell staining reagent: 
- HBSS: 500 µL
- Hoechst 33342 (10 mg/mL): 1 µL
- NucGreen™ Dead 488 ReadyProbes™ Reagent: 1 drop
Perform bronchial biopsy at the desired level of the airways (to be performed by a medical doctor) 

Equipment
Biopsy forceps
NAME
Medi-Globe
BRAND
GBF-21-18-120
SKU

Put the biopsy in 1 mL DPBS in a well of a 6-well plate, observe aspect, and then transfer into 1 mL of ice-cold dissociation buffer in a 1.5 mL eppendorf tube. Use wide-bore 1 mL pipet tip for all biopsy transfers.
Amount1 mL
Note
PREPARATION OF DISSOCIATION MIX (Fresh at each experiment)
Ingredients:
- PBS
- Protease from Bacillus Licheniformis (100 mg/mL stock solution in PBS)
- EDTA 10 mM
For 1 mL of dissociation mix add:
- 850 microlitres of PBS
- 100 microlitres of protease (Final concentration:10 mg/mL)
- 50 microlitres of EDTA (Final concentration: 0.5 mM)
Temperature4 °C

Expected result








If transportation or storage is necessary: place tube on ice or in polystyrene box containing ice packs. Biopsies can be stored for 60 min in dissociation buffer.
Carefully remove biopsy from the dissociation buffer, with a wide-bore 1 mL pipet tip and place in a 100 mm petri dish, taking as little dissociation buffer as possible. Mince with a scalpel equipped of a 10 blade. Drag the biopsy out of the liquid and mince very carefully into the smallest possible pieces. With a wide-bore pipet tip, transfer back the minced biopsy with a small volume of dissociation buffer. Rince the petri dish with dissociation buffer, at the location of biopsy mincing to recover as many cells as possible.
Incubate cells on ice for 90 to 120 min after mincing, with gentle trituration with needles 5 times every 5 min. Use needle with decreasing sizes from 21G to 23G.

Duration01:30:00 Incubation
Duration00:05:00 Trituration
Temperature4 °C
Inactivate protease by adding 200 µL of Inactivation buffer (HBSS containing 2% BSA)
Amount200 µL Inactivation buffer
Note
Prepare Inactivation buffer:
HBSS : 40 mL
10% BSA stock: 10 mL
Spin at 400g for 5 min at 4°C
Discard supernatant leaving 10 µL of residual liquid on the pellet.
Resuspend in 100 µL of wash buffer (HBSS + 1% BSA)
Amount100 µL wash buffer
Note
Prepare Wash buffer:
HBSS : 20 mL
HBSS/BSA 2%: 20 mL

Observe cells under an inverted microscope to evaluate red blood cells (RBC) content. 
RBC content is better evaluated using an automated cell counter such as Countess, after addition of Hoechst 33342 to an aliquot of the cell suspension to discriminate nucleated cells from non-nucleated cells.

If RBC content iis lower than 50%, go directly to step 18.
Perform RBC lysis: add 900 µL of Ammonium Chloride 0.8% to 100 µL of cell suspension (9 volumes). 
ReagentAmmonium Chloride Solution 100 mL VWR International (Avantor)Catalog #7800
Amount900 µL Ammonium Chloride 9 vol. for 1 cell vol.
Incubate on ice for 10 min.
Duration00:10:00 RBC lysis
Temperature4 °C
 Add 200 µL of Inactivation buffer
Amount200 µL Inactivation buffer
Spin at 400g for 5 min at 4°C
Discard supernatant leaving 10 µL of residual liquid on the pellet.
Resuspend in 100 µL of wash buffer and monitor correct RBC lysis under microscope
Amount100 µL wash buffer
Add 1 mL of wash buffer
Amount1 mL wash buffer
Spin at 400g for 5 min at 4°C
Discard supernatant leaving 10 µL of residual liquid on the pellet.
Resuspend in 500 µL of wash buffer
Amount500 µL wash buffer
Filter cell suspension through Flowmi cell strainer
ReagentFlowmi cell strainerVWR International (Avantor)Catalog #H13680-0040
https://www.youtube.com/watch?v=taS1BuTnvds
Add 500 µL of wash buffer to filtered cells.
Amount500 µL wash buffer
Discard supernatant leaving 10 µL of residual liquid on the pellet.
Resuspend in 40 µL of resuspension buffer (HBSS + 0.05% BSA).

Amount40 µL Resuspension buffer

Note

Prepare Resuspension buffer:
HBSS : 39 mL
HBSS/BSA 2%: 1 mL


Mix 10 µL of cells with 10 µL of cell counting solution (HBSS with 20 µg/mL Hoechst 33342 and NucGreen). Incubate for 1 min at room temperature.
ReagentHoechst 33342, Trihydrochloride, Trihydrate - 10 mg/mL Solution in WaterVWR International (Avantor)Catalog #H3570
ReagentNucGreen™ Dead 488 ReadyProbes™ ReagentVWR International (Avantor)Catalog #R37109
Note
Preparation of cell staining reagent:
- HBSS: 500 µL
- Hoechst 33342 (10 mg/mL): 1 µL
- NucGreen™ Dead 488 ReadyProbes™ Reagent: 1 drop
Duration00:01:00
Count with Countess automated cell counter using both sides of chambers. Monitor the percentage of nucleated cells (Hoechst +) and dead cells (GFP+). 

Equipment
new equipment
NAME
Thermo Fisher Scientific
BRAND
AMQAF1000
SKU
Countess™ II FL Automated Cell Counter with Dapi and GFP cubes
SPECIFICATIONS

Expected result
Countess GFP image after NucGreen and Hoescht33342 staining
Countess Dapi image after NucGreen and Hoescht33342 staining
Countess report after NucGreen and Hoescht33342 staining
Countess report after NucGreen and Hoescht33342 staining



Expected result
Floid image after NucGreen and Hoescht33342 staining



Adjust concentration to a range of 700 to 1000 cells/µL (with wash buffer) for 10X Chromium. Monitor final cell concentration.