May 18, 2022
Version 2
Processing of pediatric bronchoalveolar lavage samples for single cell analysis V.2
- Shivanthan Shanthikumar1,
- Richard Saffery1,
- Sarath C. Ranganathan1,
- Melanie R Neeland1
- 1Murdoch Children's Research Institute
Protocol Citation: Shivanthan Shanthikumar, Richard Saffery, Sarath C. Ranganathan, Melanie R Neeland 2022. Processing of pediatric bronchoalveolar lavage samples for single cell analysis . protocols.io https://dx.doi.org/10.17504/protocols.io.36wgq4b9ovk5/v2Version created by Melanie R Neeland
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: May 18, 2022
Last Modified: April 25, 2024
Protocol Integer ID: 62786
Keywords: single cell analysis, BAL, respiratory, pediatric, lung, flow cytometry
Abstract
This protocol describes the collection, processing, cryopreservation and thawing of pediatric bronchoalveolar lavage (BAL) samples for downstream single cell analysis (including flow cytometry, cell sorting, and single cell transcriptomics).
Guidelines
This is an experimental protocol for processing of bronchoalveolar lavage samples collected from children. Sample collection must have and be compliant with Human Ethics Committee approval.
For guidelines on how to safely perform bronchoscopy and lavage in children, please see:
CITATION
Materials
RPMI-1640Sigma AldrichCatalog #R5886
Fetal Bovine Serum
1X PBS (Phosphate-buffered saline )
DMSO (dimethyl sulfoxide)Sigma AldrichCatalog #D8418
conical tubes, 50ml
conical tubes, 15ml
Benzonase® NucleaseMerck MilliporeCatalog #E1014-25KU
Corning® cell strainerCorningCatalog #CLS431751-50EA
Safety warnings
Human samples should be processed in a laboratory with appropriate biosafety infrastructure and procedures.
COLLECTION OF BAL.
COLLECTION OF BAL.
After obtaining informed consent from family and/or patient, obtain any excess BAL fluid collected at the time of clinically indicated bronchoscopy and lavage.
For guidelines on how to safely perform bronchoscopy and lavage in children, please see:
CITATION
BAL samples must be placed on ice and processed in the laboratory within 30 minutes -1 hour of the procedure.
PROCESSING OF BAL TO RECOVER SINGLE CELLS.
PROCESSING OF BAL TO RECOVER SINGLE CELLS.
30m
30m
Centrifuge BAL samples at 300 x g, 4°C, 00:10:00 .
10m
Remove supernatant and resuspend cell pellet in 10mL of pre-chilled RPMI supplemented with 2% heat-inactivated fetal calf serum (herein referred to as RPMI 2% FCS).
Note
Cell-free BAL supernatant can be stored at -80 °C for future proteomic analysis (e.g. quantification of cytokines)
Filter cell suspension through a 70-120µm cell strainer and centrifuge filtered cell suspension 300 x g, 4°C, 00:10:00 .
Note
In some cases, BAL samples may require a second filtering step to remove additional debris
10m
Discard supernatant and resuspend cell suspension in 2mL chilled RPMI 2% FCS. Remove 10µL for cell counting. Top up the cell suspension to 10mL RPMI 2% FCS and centrifuge 300 x g, 4°C, 00:10:00 while performing cell count.
Note
Cell counting can be performed manually using a haemocytometer, or using an automated cell counter (although the accuracy of some automated cell counters is limited by their inability to count large alveolar macrophages)
10m
OPTION: Workflow for single cell analysis of fresh BAL samples.
OPTION: Workflow for single cell analysis of fresh BAL samples.
If samples will be cryopreserved for storage, proceed to step 9 of this protocol.
If samples will be processed for immediate analysis (e.g. flow cytometry, cell sorting, or sc-RNAseq of live single cells) the following steps can be performed:
Resuspend cell suspension in chilled staining buffer for fixable viability staining according to manufacturers' instructions (e.g. the LIVE/DEAD™ Fixable Near-IR Stain from Invitrogen/ThermoFisher).
For a protocol detailing flow cytometry analysis of fresh BAL samples, please see our article:
CITATION
CRYOPRESERVATION OF BAL.
CRYOPRESERVATION OF BAL.
Discard supernatant and resuspend cells at a ratio of 1:1 in chilled RPMI 2% FCS and freeze solution (heat-inactivated FCS + 15% DMSO) such that cells are frozen between 1-10 million cells/mL.
Note
This step should be done on ice. Add freeze solution to cell suspension in a drop-by-drop manner.
Immediately place cryogenic vials into an isopropanol freezing container (e.g. Nalgene® Mr. Frosty) and transfer to -80 °C overnight.
For long term storage, transfer the vials of frozen BAL cells to liquid nitrogen.
THAWING OF CRYOPRESERVED BAL FOR SINGLE CELL ANALYSIS.
THAWING OF CRYOPRESERVED BAL FOR SINGLE CELL ANALYSIS.
12m
12m
Warm thaw media (RPMI + 10% heat-inactivated FCS + 25U/mL Benzonase) to 37 °C in a water bath.
Note
For every sample to be thawed, place 8mL of warmed thaw media into a 15mL tube.
Remove cryopreserved BAL samples from liquid nitrogen and keep on dry ice for transport to the laboratory.
Place cryovials into the water bath for cell thawing, approximately 00:02:00 .
2m
Using a pasteur pipette, transfer cells from cryovial into the 15mL tube containing warmed thaw media.
Rinse cryovial with 1mL warmed thaw media to recover any remaining cells and transfer to the 15mL tube.
Centrifuge the cell suspension at 300 x g, 00:10:00 at room temperature.
10m
Discard the supernatant and resuspend the cell pellet in 1mL RPMI 2%FCS for cell counting, followed by a final wash in 10mL RPMI 2%FCS and centrifuge at 300 x g, 00:10:00 at room temperature.
10m
Once the supernatant has been discarded, the cells are now ready to be resuspended at the required dilution for the first steps in your single cell experiment.
Expected result
Following cryopreservation and thaw of n=21 pediatric BAL samples as described in this protocol, we achieved a median viability of 76.1% (range 61-90.7%) (determined by live/dead staining using flow cytometry).
Expected result
Cryopreservation and thaw of BAL samples will result in the loss of some granulocyte populations. For a fresh vs thaw comparison of the immune cell profile of BAL, see our protocol published here:
CITATION
PREPARATION OF CELLS FOR FLOW CYTOMETRY
PREPARATION OF CELLS FOR FLOW CYTOMETRY
45m
45m
Resuspend cell suspension for fixable viability staining according to manufacturers' instructions (e.g. the LIVE/DEAD™ Fixable Near-IR Stain from Invitrogen/ThermoFisher).
Following the required incubation, stop the reaction by the addition of 1mL staining buffer (2% heat-inactivated FCS in PBMS 2mM EDTA, herein referred to as FACS buffer) and centrifuge at 400 x g, 4°C, 00:05:00 .
5m
Resuspend cells in human FC-block according to manufacturers’ instructions for 00:05:00 at room temperature.
5m
Add required antibody cocktail made up at 2X concentration 1:1 with the cells and incubate for 00:30:00 on ice. For examples of relevant antibody stains for pediatric lung samples, please see our published work below.
CITATION
30m
Following staining, wash cells with 2mL FACS buffer and centrifuge at 400 x g, 4°C, 00:05:00 and resuspend cells in >100 µL for acquisition on a flow cytometer.
5m
Citations
Faro A, Wood RE, Schechter MS, Leong AB, Wittkugel E, Abode K, Chmiel JF, Daines C, Davis S, Eber E, Huddleston C, Kilbaugh T, Kurland G, Midulla F, Molter D, Montgomery GS, Retsch-Bogart G, Rutter MJ, Visner G, Walczak SA, Ferkol TW, Michelson PH, American Thoracic Society Ad Hoc Committee on Flexible Airway Endoscopy in Children.. Official American Thoracic Society technical standards: flexible airway endoscopy in children.
https://doi.org/10.1164/rccm.201503-0474STStep 2
Faro A, Wood RE, Schechter MS, Leong AB, Wittkugel E, Abode K, Chmiel JF, Daines C, Davis S, Eber E, Huddleston C, Kilbaugh T, Kurland G, Midulla F, Molter D, Montgomery GS, Retsch-Bogart G, Rutter MJ, Visner G, Walczak SA, Ferkol TW, Michelson PH, American Thoracic Society Ad Hoc Committee on Flexible Airway Endoscopy in Children.. Official American Thoracic Society technical standards: flexible airway endoscopy in children.
https://doi.org/10.1164/rccm.201503-0474STStep 22
Shanthikumar S, Ranganathan SC, Saffery R, Neeland MR. Mapping Pulmonary and Systemic Inflammation in Preschool Aged Children With Cystic Fibrosis.
https://doi.org/10.3389/fimmu.2021.733217Step 8.2
Shanthikumar S, Ranganathan SC, Saffery R, Neeland MR. Mapping Pulmonary and Systemic Inflammation in Preschool Aged Children With Cystic Fibrosis.
https://doi.org/10.3389/fimmu.2021.733217