Feb 13, 2019
Version 2
Cell dissociation from nasal and bronchial brushings with cold-active protease for single-cell RNA-seq V.2
- 1Universite Cote d’Azur, CNRS, IPMC, 06560 Valbonne, France
- Human Cell Atlas Method Development Community
Protocol Citation: Laure-Emmanuelle Zaragosi, Pascal Barbry 2019. Cell dissociation from nasal and bronchial brushings with cold-active protease for single-cell RNA-seq. protocols.io https://dx.doi.org/10.17504/protocols.io.qubdwsn
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: June 08, 2018
Last Modified: February 13, 2019
Protocol Integer ID: 12899
Keywords: brushing, bronchail epithelium, nasal 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 nasal epithelium brushings.
Cell dissociation is performed at 4°C to avoid gene expression alterations and maximize viability.
The typical cell number recovery is 200 000 - 300 000 for one brushing.
Cell suspensions are suitable for single-cell RNA-sequencing protocols.
Guidelines
Storage Conditions of Reagents
| Reagent | Storage Condition | |
| HBSS | 4°C | |
| Hypothermosol | 4°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 brushing of the epithelium of the nasal cavity
2. Dissociation: triturate on ice or store on ice without trituration
3. Remove red blood cells if necessary
4. Prepare cells for Chromium/DropSeq
All steps should be performed on ice or at 4°C
Materials
MATERIALS
EDTA
23G NeedlesCatalog #4657667
Protease from Bacillus Licheniformis Merck MilliporeSigma (Sigma-Aldrich)Catalog #P5380
HypoThermosol® FRS 100 mL
STEMCELL Technologies Inc.Catalog #7935
Quick-Read 10 Chamber SlideGlobe ScientificCatalog #3805
Countess™ Cell Counting Chamber SlidesCatalog #C10314
DPBS no calcium, no magnesiumInvitrogen - Thermo FisherCatalog #14190136
21G needleVWR International (Avantor)Catalog #BD-305165
HBSSGibco - Thermo Fisher ScientificCatalog #14060040
STEP MATERIALS
Quick-Read 10 Chamber SlideGlobe ScientificCatalog #3805
Flowmi cell strainerCatalog #H13680-0040
Hoechst 33342, Trihydrochloride, Trihydrate - 10 mg/mL Solution in WaterThermo Fisher ScientificCatalog #H3570
NucGreen™ Dead 488 ReadyProbes™ ReagentThermo Fisher ScientificCatalog #R37109
Ammonium Chloride Solution 100 mL
STEMCELL Technologies Inc.Catalog #7800
Protocol materials
DPBS no calcium, no magnesiumInvitrogen - Thermo FisherCatalog #14190136
23G NeedlesCatalog #4657667
Hoechst 33342, Trihydrochloride, Trihydrate - 10 mg/mL Solution in WaterThermo Fisher ScientificCatalog #H3570
21G needleVWR International (Avantor)Catalog #BD-305165
Countess™ Cell Counting Chamber SlidesCatalog #C10314
EDTA
Ammonium Chloride Solution 100 mL
STEMCELL Technologies Inc.Catalog #7800
Protease from Bacillus Licheniformis Merck MilliporeSigma (Sigma-Aldrich)Catalog #P5380
HypoThermosol® FRS 100 mL
STEMCELL Technologies Inc.Catalog #7935
Quick-Read 10 Chamber SlideGlobe ScientificCatalog #3805
Flowmi cell strainerCatalog #H13680-0040
NucGreen™ Dead 488 ReadyProbes™ ReagentThermo Fisher ScientificCatalog #R37109
HBSSGibco - Thermo Fisher ScientificCatalog #14060040
Quick-Read 10 Chamber SlideGlobe ScientificCatalog #3805
Quick-Read 10 Chamber SlideGlobe ScientificCatalog #3805
Ammonium Chloride Solution 100 mL
STEMCELL Technologies Inc.Catalog #7800
Hoechst 33342, Trihydrochloride, Trihydrate - 10 mg/mL Solution in WaterThermo Fisher ScientificCatalog #H3570
NucGreen™ Dead 488 ReadyProbes™ ReagentThermo Fisher ScientificCatalog #R37109
Flowmi cell strainerCatalog #H13680-0040
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 | |
| 850 | Hypothermosol | 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 nasal brush biopsy in the nasal cavity in the inferior nasal conca zone (UBERON_0005922) (to be performed by a medical doctor)
Equipment
new equipment
NAME
Medi-Globe
BRAND
GCB-02-18-120
SKU
2.0 mm cytology brush
SPECIFICATIONS
Cut the cytology brush and place it in a 5 mL eppendorf tube containing 1 mL of ice-cold dissociation buffer.
1 mL
Note
PREPARATION OF DISSOCIATION MIX (Fresh at each experiment)
Ingredients:
- Hypothermosol
- Protease from Bacillus Licheniformis (100 mg/mL stock solution in PBS)
- EDTA 10 mM
For 1 mL of dissociation mix add:
- 850 microlitres of Hypothermosol
- 100 microlitres of protease (Final concentration:10 mg/mL)
- 50 microlitres of EDTA (Final concentration: 0.5 mM)
4 °C
If cells are processed directly after brushing, use DPBS instead of hypothermosol and go directly to step 4.
If transportation or storage is necessary: place tube on ice or in polystyrene box containing ice packs. Brushings can be stored for 60 min in dissociation buffer.
Shake the brush into the buffer to put cells in suspension then spin the tube for 2 min at 150g to remove residual cells from the brush.
Discard the cytology brush and observe cells under an inverted microscope.
Quick-Read 10 Chamber SlideGlobe ScientificCatalog #3805
Expected result
Incubate cells on ice for 30 min, with gentle trituration with needles 5 times every 5 min. Use needle with decreasing sizes from 21G to 23G.
If storage and/or transportation has been performed for at least 60 min, skip the incubation step, only perform trituration steps.
00:30:00 Incubation
00:05:00 Trituration
Expected result
4 °C
Inactivate protease by adding 200 µL of Inactivation buffer (HBSS containing 2% BSA)
200 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 200 µL of wash buffer (HBSS + 1% BSA)
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.
Expected result
If RBC content iis lower than 50%, go directly to step 18.
Perform RBC lysis: add 1.8 mL of Ammonium Chloride 0.8% to 200 µL of cell suspension (9 volumes), transfer in a 5 mL eppendorf.
Ammonium Chloride Solution 100 mL
STEMCELL Technologies Inc.Catalog #7800
2.25 mL Ammonium Chloride 9 vol. for 1 cell vol.
Incubate on ice for 10 min.
00:10:00 RBC lysis
4 °C
Add 400 µL of Inactivation buffer
2 mL Inactivation buffer
Spin at 400g for 5 min at 4°C
Discard supernatant leaving 10 µL of residual liquid on the pellet.
Resuspend in 250 µL of wash buffer and monitor correct RBC lysis
250 µL wash buffer
Add 1 mL of wash buffer
1 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
500 µL wash buffer
Filter cell suspension through Flowmi cell strainer
Flowmi cell strainerCatalog #H13680-0040
Add 500 µL of wash buffer to filtered cells.
500 µL wash buffer
Discard supernatant leaving 10 µL of residual liquid on the pellet.
Resuspend in 50 µL of resuspension buffer (HBSS + 0.05% BSA).
50 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.
Hoechst 33342, Trihydrochloride, Trihydrate - 10 mg/mL Solution in WaterThermo Fisher ScientificCatalog #H3570
NucGreen™ Dead 488 ReadyProbes™ ReagentThermo Fisher ScientificCatalog #R37109
Note
Preparation of cell staining reagent:
- HBSS: 500 µL
- Hoechst 33342 (10 mg/mL): 1 µL
- NucGreen™ Dead 488 ReadyProbes™ Reagent: 1 drop
00:01:00
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 report after NucGreen and Hoescht33342 staining
Expected result
Countess bright field image after NucGreen and Hoescht33342 staining
Expected result
Countess Dapi image after NucGreen and Hoescht33342 staining
Floid microscope 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.