May 08, 2024
Processing of pediatric bronchoalveolar lavage samples for single cell analysis v 3
- 1Murdoch Children's Research Institute
Protocol Citation: Shivanthan Shanthikumar, Liam Gubbels, Melanie R Neeland 2024. Processing of pediatric bronchoalveolar lavage samples for single cell analysis v 3. protocols.io https://dx.doi.org/10.17504/protocols.io.6qpvr8ox2lmk/v1
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: April 09, 2024
Last Modified: May 08, 2024
Protocol Integer ID: 97934
Keywords: single cell analysis, BAL, respiratory, pediatric, lung, flow cytometry
Funders Acknowledgement:
Chan Zuckerberg Initiative
Grant ID: 2021-237883
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 BRONCHOALVEOLAR LAVAGE (BAL)
COLLECTION OF BRONCHOALVEOLAR LAVAGE (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 SINGLE CELL SUSPENSION
PROCESSING OF BAL TO SINGLE CELL SUSPENSION
30m
Centrifuge BAL samples at 300 x g, 4°C, 00:07:00 .
7m
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, 00:07:00 .
Note
In some cases, BAL samples may require a second filtering step to remove additional debris
7m
Discard supernatant and resuspend cell pellet in 3 mL RPMI 2% FCS.
Prepare cell suspension for cell counting. Here, we use AO/PI and the LUNA FL counter. Remove 18 µL for cell counting into a microcentrifuge tube. Add 2µL of AO/PI to the count tube and mix well.
Load 10µL of stained cells onto a Luna fluorescent counting slide and count. Record viability, total cell count, and live cell count.
Note
Cell counting can be performed manually using a haemocytometer, or using other automated cell counters.
If choosing to run flow cytometry or other single cell assays on fresh cells, here is where you can allocate the required number of cells for downstream processing. For flow cytometry, described below, we allocate 200-300,000 cells prior to proceeding to cryopreservation for remaining cells.
CRYOPRESERVATION OF BAL MONONUCLEAR CELLS
CRYOPRESERVATION OF BAL MONONUCLEAR CELLS
Top up remaining cell suspension to 8mL with RPMI 2% FCS.
Fill a 15mL tube with 2mL of Ficoll plaque plus and layer the cell suspension onto the surface of the Ficoll solution.
Note
We choose to use a Ficoll gradient for cryopreservation. This is to remove fragile granulocytes that may affect viability of the sample upon thaw. We observe higher viability upon thaw following Ficoll preparation when compared to cryopreservation of whole BAL.
Note
Layer the cell suspension slowly to prevent the Ficoll solution from mixing with the cells.
Centrifuge the layered cell suspension at 400 x g, Room temperature, 00:30:00 , 4 Acceleration and NO brake .
30m
Once the spin is complete, carefully aspirate the mononuclear layer at the interface between the RPMI 2% FCS and the Ficoll solution into a new 15mL tube. Top up the cell suspension to 10mL with RPMI 2% FCS and centrifuge 400 x g, 4°C, 00:05:00 .
Note
When collecting the cells, try to avoid Ficoll solution as much as possible.
5m
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. Transfer cells to cryogenic vial.
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 vials to liquid nitrogen.
PREPARATION OF FRESH CELLS FOR FLOW CYTOMETRY
PREPARATION OF FRESH CELLS FOR FLOW CYTOMETRY
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 25µL of FC-block for 00:05:00 at Room temperature .
5m
The next steps will depend on the requirements for your specific panel. As an example, we have attached our 17-plex spectral cytometry panel that we routinely use on paediatric airway samples, as well as a publication describing the application of this panel. All of the following steps are related to this panel.
nasal_bronchial_BAL_panel.pdf114KB CITATION
Add 25µL of antibody cocktail made up at 2X concentration and incubate for 00:30:00 On ice .
30m
Following staining, wash cells with 2mL FACS buffer, centrifuge at 400 x g, 4°C, 00:05:00 and resuspend cells in 200µL FACS buffer for acquisition on a flow cytometer (here, a Cytek 5L Aurora).
5m
Immediately before running the sample, filter the cell suspension through a 35 µm cell strainer ( Falcon‱ 352235).
OPTION: THAWING OF CRYOPRESERVED BAL CELLS FOR SINGLE CELL ANALYSIS.
OPTION: THAWING OF CRYOPRESERVED BAL CELLS FOR SINGLE CELL ANALYSIS.
12m
If it is not possible to run single cell assays on fresh cells on the day of bronchoscopy, herein is a protocol for single cell analysis on cryopreserved and thawed BAL cells.
Noting this will not enable efficient analysis of granulocytes, which were removed during the Ficoll gradient steps above.
Granulocytes are unlikely to survive a cryopreservation and thaw process even if whole BAL was cryopreserved.
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, 00:07:00 at room temperature.
7m
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, 00:07:00 at room temperature.
7m
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.
Citations
Step 19
Neeland MR, Gubbels L, Wong ATC, Walker H, Ranganathan SC, Shanthikumar S. Pulmonary immune profiling reveals common inflammatory endotypes of childhood wheeze and suppurative lung disease.
https://doi.org/pii:S1933-0219(24)00020-5.10.1016/j.mucimm.2024.03.001