Nov 09, 2023
Preparation of a Single Cell Suspension from Bronchoalevolar Lavage
- Steven B. Wells1,
- Peter A. Szabo2,
- Basak Ural2
- 1Department of Systems Biology, Columbia University Irving Medical Center, New York, NY 10032, USA;
- 2Department of Microbiology and Immunology, Columbia University Irving Medical Center, New York, NY 10032, USA
- Columbia
Protocol Citation: Steven B. Wells, Peter A. Szabo, Basak Ural 2023. Preparation of a Single Cell Suspension from Bronchoalevolar Lavage. protocols.io https://dx.doi.org/10.17504/protocols.io.bwrjpd4n
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: July 20, 2021
Last Modified: November 09, 2023
Protocol Integer ID: 51723
Keywords: Lung, BAL, Airway, CD45, Lymphocytes, Myeloid, Isolation, Density gradient, Ficoll, Immune, 10x, scRNAseq, Flow cytometry, Leukocyte, Single cell suspension, T cell,
Abstract
This protocol describes a method for the isolation of the immune cells, structural and epithelial cells, and progenitors from lavage fluid collected from human lung. By providing defined media formulations, volumes at each step, and a defined dilution factor for density centrifugation, it yields consistent single-cell suspensions across samples.
Attachments
dzhjbk587.pdf
88KB
Materials
Materials:
- Syringes Fitted with Luer Lock Valve (509353)Millipore SigmaCatalog #509639 25mL Syringe
- BD Syringes without Needle 50 mLFisher ScientificCatalog #13-689-8
- BD Angiocath Peripheral IV Catheter 12G x 76mm (10)BD BiosciencesCatalog #382277
- Benzonase nucleaseSigma AldrichCatalog #E1014-5KU
- 3-Way StopcocksBio-rad LaboratoriesCatalog #7328103
- DPBS no calcium no magnesiumThermo Fisher ScientificCatalog #14190144
- Penicillin-Streptomycin-Glutamine (100X)Thermo FisherCatalog #10378016
- Thermo Scientific™ Nunc™ 50mL Conical Sterile Polypropylene Centrifuge TubesFisher ScientificCatalog #12-565-271
- Gibco™ IMDM (Iscoves Modified Dulbeccos Medium)Fisher ScientificCatalog #12-440-053
- Gibco™ Fetal Bovine Serum qualified AustraliaFisher ScientificCatalog #10-099-141
- UltraPure™ 0.5 M EDTA pH 8.0Thermo Fisher ScientificCatalog #15575020 )
- Thomas ScientificSupplier Diversity Partner Cell Strainer 100um Yellow Sterile Individually WrapFisher ScientificCatalog #50-146-1428
- Ficoll-Paque™ PLUS MediaFisher ScientificCatalog #45-001-749
- Mr. Frosty™ Freezing ContainerFisher ScientificCatalog #5100-0001
- CryoStor CS10 100MLFisher ScientificCatalog #NC9930384
- Corning™ Externally Threaded Cryogenic VialsFisher ScientificCatalog #09-761-71
- 5mL Falcon™ Round-Bottom Polypropylene Test TubesFisher ScientificCatalog #14-959-11A
- Solution 13 AO – DAPIChemometecCatalog #910-3013
- NC-Slide A8™ box with 25 SlidesChemometecCatalog #942-0003
- Falcon™ Plastic Disposable Transfer PipetsFisher ScientificCatalog #1368050
Equipment
- Multi-Axle-Rotating Mixer/Shaker with Temperature Control
- Centrifuge
- Cell Counter - NC-3000
- Surgical scissors
- Scale
Preparing Mediums and Buffers
Preparing Mediums and Buffers
Create the following IMDM-FBS-PSQ Media in a 500 mL bottle of IMDM by using the table below:
| A | B | C | D | |
| Component | Volume (mL) | Starting Conc. | Final Conc.* | |
| IMDM | 500 | - | - | |
| Penicillin-Streptomycin-Glutamine | 5 | 100X | 1X | |
| FBS | 50 | 100% | 10% |
Table 1.
*Final Concentration is approximate.
Create the following DPBS-FBS-EDTA Solution in a bottle of DPBS without calcium and magnesium by using the table below:
| A | B | C | D | |
| Component | Volume (mL) | Starting Conc. | Final Conc.* | |
| DPBS | 500 | - | - | |
| FBS | 25 | 100% | 5% | |
| EDTA | 50 | 0.5M | 1mM |
Table 2.
*Final Concentration is approximate.
Performing the Lavage
Performing the Lavage
Identify, and using a scissors, make an incision in one of the secondary bronchi that connects to the lower lobes of the left lung.
Insert the catheter about 5 to 10 centimeters into the incision, remove the needle and attach a 3- way Stopper to the catheter.
Fill a 50 mL syringe with PBS and connect to the 3-way Stopper.
Slowly inject 25 mL of cold PBS into the lungs. Watch the lungs inflate, and do not overinflate.
Attach an empty 25 mL syringe to the final spot of the 3-way Stopper.
Collect about 10 mL of BAL fluid (BALF) from lungs.
Repeat the previous steps until 50 mL of PBS is injected into the lungs and at least 25 mL to 50 mL of BALF is collected.
Processing the BALF
Processing the BALF
5m
5m
Spin for 00:05:00 at 400 x g at 4 °C , remove and save the supernatant in 2 mL cyrovials – record supernatant volume saved below:
_________ mL
5m
Resuspend the cell pellet in 10 mL of IMDM, add 10 µL of benzonase to the BALF and at 37 °C for 00:30:00 .
30m
Add 40 mL of IMDM (NO ADDITIVES) to the cell suspension, spike in 0.500 mL of EDTA 0.5 Molarity (M) 8.0 .
Ficoll-Paque
Ficoll-Paque
40m
40m
Filter the cell suspension through a 100 micromolar (µM) cell strainer.
In two 50 mL tubes, layer 25 mL of cell suspension on top of 15 mL of Ficoll-Paque Media PLUS.
Spin for 00:20:00 , 1200 x g at 20 °C with 4 acceleration and 0 brake, evenly distribute the tubes across the entire rotor to prevent wobbling (use all four buckets if possible as opposed to just two).
20m
Remove the mononuclear cell layer from both tubes with a transfer pipet and combine in one 50 mL tube. Add cold DPBS-FBS-EDTA Solution to a final volume of 50 mL and centrifuge the cell suspension for 00:10:00 at 400 x g , 4 °C .
10m
Remove the supernatant and re-suspend the cell pellet in 50 mL cold DPBS-FBS-EDTA Solution and centrifuge the cell suspension for 00:10:00 at 120 x g , 4 °C .
10m
Remove the supernatant and re-suspend the cell pellet in cold 10 mL IMDM-FBS-PSQ Media.
Cell Count
Cell Count
Count cells, and viability by using the NC-3000 cell counter. Calculate total viable cells and record below:
cell number: __________ cells/mL, __________ %viable
final volume: __________ mL
𝑐𝑒𝑙𝑙 𝑛𝑢𝑚𝑏𝑒𝑟 (𝑐𝑒𝑙𝑙𝑠/𝑚𝐿) ∗ 𝑣𝑖𝑎𝑏𝑖𝑙𝑖𝑡𝑦(%) ∗ 𝑓𝑖𝑛𝑎𝑙 𝑣𝑜𝑙𝑢𝑚𝑒(𝑚𝐿) = 𝑡𝑜𝑡𝑎𝑙 𝑣𝑖𝑎𝑏𝑙𝑒 𝑐𝑒𝑙𝑙𝑠
Total Viable Cells: __________
Freeze-down and QC
Freeze-down and QC
(Optional QC) Aliquot 2 x 106 cells to a 5mL Falcon tube and place on ice for subsequent flow cytometric analysis.
Aliquot cells for analysis or experimentation, and then freeze down cells in up to 2 x 107 aliquots using Cryostor CS10 Medium, a Mr. Frosty, and a -80 °C freezer (1 mL -1.5 mL aliquots, round down to the nearest 30 million cells and discard/freeze/use any left over cells). Record the number of vials frozen: __________.