TITLE: Processing of pediatric bronchoalveolar lavage samples for single cell analysis
AUTHORS: Shivanthan Shanthikumar, Richard Saffery, Sarath C. Ranganathan, Melanie Neeland

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">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).</div></div>

[STEPS]
?. [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.
?. [PROCESSING OF BAL TO RECOVER SINGLE CELLS.]
Centrifuge BAL samples at  .
Centrifuge: 300 34, 10 min, 4 10
?. [PROCESSING OF BAL TO RECOVER SINGLE CELLS.]
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).
Cell-free BAL supernatant can be stored at    for future proteomic analysis (e.g. quantification of cytokines)
?. [PROCESSING OF BAL TO RECOVER SINGLE CELLS.]
Filter cell suspension through a 70-120µm cell strainer and centrifuge filtered cell suspension .
Centrifuge: 300 34, 10 min, 4 10
In some cases, BAL samples may require a second filtering step to remove additional debris
?. [PROCESSING OF BAL TO RECOVER SINGLE CELLS.]
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  while performing cell count.
Centrifuge: 300 34, 10 min, 4 10
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)
?. [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:
?. [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.
This step should be done on ice. Add freeze solution to cell suspension in a drop-by-drop manner.
?. [CRYOPRESERVATION OF BAL.]
Immediately place cryogenic vials into an isopropanol freezing container (e.g. Nalgene® Mr. Frosty) and transfer to  overnight.
-80 °C
?. [CRYOPRESERVATION OF BAL.]
For long term storage, transfer the vials of frozen BAL cells to liquid nitrogen.
?. [OPTION: Workflow for single cell analysis of fresh BAL samples. ]
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).
?. [OPTION: Workflow for single cell analysis of fresh BAL samples. ]
For a protocol detailing flow cytometry analysis of fresh BAL samples, please see our article:
Following processing of n=11 pediatric BAL samples as described in this protocol, we achieved a median viability of 78.9% (range 72-88.8%) (determined by live/dead staining using flow cytometry).
?. [THAWING OF CRYOPRESERVED BAL FOR SINGLE CELL ANALYSIS.]
Warm thaw media (RPMI + 10% heat-inactivated FCS + 25U/mL Benzonase) to   in a water bath.
37 °C
For every sample to be thawed, place 8mL of warmed thaw media into a 15mL tube.
?. [THAWING OF CRYOPRESERVED BAL FOR SINGLE CELL ANALYSIS.]
Remove cryopreserved BAL samples from liquid nitrogen and keep on dry ice for transport to the laboratory.
?. [THAWING OF CRYOPRESERVED BAL FOR SINGLE CELL ANALYSIS.]
Place cryovials into the water bath for cell thawing, approximately .
?. [THAWING OF CRYOPRESERVED BAL FOR SINGLE CELL ANALYSIS.]
Using a pasteur pipette, transfer cells from cryovial into the 15mL tube containing warmed thaw media.
?. [THAWING OF CRYOPRESERVED BAL FOR SINGLE CELL ANALYSIS.]
Rinse cryovial with 1mL warmed thaw media to recover any remaining cells and transfer to the 15mL tube.
?. [THAWING OF CRYOPRESERVED BAL FOR SINGLE CELL ANALYSIS.]
Centrifuge the cell suspension at  at room temperature.
Centrifuge: 300 34, 10 min
?. [THAWING OF CRYOPRESERVED BAL FOR SINGLE CELL ANALYSIS.]
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.
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).
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:
?. [OPTION: Flow cytometry analysis of thawed BAL.]
For flow cytometry analysis of cryopreserved BAL, please see our protocol published here:
?. [COLLECTION OF BAL.]
For guidelines on how to safely perform bronchoscopy and lavage in children, please see:
?. [COLLECTION OF BAL.]
BAL samples must be placed on ice and processed in the laboratory within 30 minutes -1 hour of the procedure.
?. [THAWING OF CRYOPRESERVED BAL FOR SINGLE CELL ANALYSIS.]
Discard the supernatant and resuspend the cell pellet in 10mL RPMI 2%FCS and centrifuge at  at room temperature.
Centrifuge: 300 34, 10 min