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

[DESCRIPTION]
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:

 


[STEPS]
SECTION: COLLECTION OF BAL.
1. After obtaining informed consent from family and/or patient, obtain any excess BAL fluid collected at the time of clinically indicated bronchoscopy and lavage.
SECTION: COLLECTION OF BAL.
2. For guidelines on how to safely perform bronchoscopy and lavage in children, please see:
 

SECTION: COLLECTION OF BAL.
3. BAL samples must be placed on ice and processed in the laboratory within 30 minutes -1 hour of the procedure. 
SECTION: PROCESSING OF BAL TO RECOVER SINGLE CELLS.
4. Centrifuge BAL samples at 300 x g, 10 min, 4 °C .
SECTION: PROCESSING OF BAL TO RECOVER SINGLE CELLS.
5. 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).
 


SECTION: PROCESSING OF BAL TO RECOVER SINGLE CELLS.
6. Filter cell suspension through a 70-120µm cell strainer and centrifuge filtered cell suspension 300 x g, 10 min, 4 °C.

 

SECTION: PROCESSING OF BAL TO RECOVER SINGLE CELLS.
7. 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, 10 min, 4 °C while performing cell count.
 



SECTION: OPTION: Workflow for single cell analysis of fresh BAL samples.
8. 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: 
SECTION: OPTION: Workflow for single cell analysis of fresh BAL samples.
8.1. 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). 
SECTION: OPTION: Workflow for single cell analysis of fresh BAL samples.
8.2. For a protocol detailing flow cytometry analysis of fresh BAL samples, please see our article:
 


SECTION: CRYOPRESERVATION OF BAL.
9. 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. 
 

SECTION: CRYOPRESERVATION OF BAL.
10. Immediately place cryogenic vials into an isopropanol freezing container (e.g. Nalgene® Mr. Frosty) and transfer to -80 °C overnight.

SECTION: CRYOPRESERVATION OF BAL.
11. For long term storage, transfer the vials of frozen BAL cells to liquid nitrogen.
SECTION: THAWING OF CRYOPRESERVED BAL FOR SINGLE CELL ANALYSIS.
12. Warm thaw media (RPMI + 10% heat-inactivated FCS + 25U/mL Benzonase) to 37 °C  in a water bath. 
 


SECTION: THAWING OF CRYOPRESERVED BAL FOR SINGLE CELL ANALYSIS.
13. Remove cryopreserved BAL samples from liquid nitrogen and keep on dry ice for transport to the laboratory.
SECTION: THAWING OF CRYOPRESERVED BAL FOR SINGLE CELL ANALYSIS.
14. Place cryovials into the water bath for cell thawing, approximately 2 min.

SECTION: THAWING OF CRYOPRESERVED BAL FOR SINGLE CELL ANALYSIS.
15. Using a pasteur pipette, transfer cells from cryovial into the 15mL tube containing warmed thaw media.
SECTION: THAWING OF CRYOPRESERVED BAL FOR SINGLE CELL ANALYSIS.
16. Rinse cryovial with 1mL warmed thaw media to recover any remaining cells and transfer to the 15mL tube.
SECTION: THAWING OF CRYOPRESERVED BAL FOR SINGLE CELL ANALYSIS.
17. Centrifuge the cell suspension at 300 x g, 10 min at room temperature.

SECTION: THAWING OF CRYOPRESERVED BAL FOR SINGLE CELL ANALYSIS.
18. 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, 10 min at room temperature. 

SECTION: THAWING OF CRYOPRESERVED BAL FOR SINGLE CELL ANALYSIS.
19. 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.
 

 

SECTION: PREPARATION OF CELLS FOR FLOW CYTOMETRY
20. 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, 5 min, 4 °C.   
SECTION: PREPARATION OF CELLS FOR FLOW CYTOMETRY
21. Resuspend cells in human FC-block according to manufacturers’ instructions for 5 min at room temperature.

SECTION: PREPARATION OF CELLS FOR FLOW CYTOMETRY
22. Add required antibody cocktail made up at 2X concentration 1:1 with the cells and incubate for 30 min  on ice. For examples of relevant antibody stains for pediatric lung samples, please see our published work below.

 

SECTION: PREPARATION OF CELLS FOR FLOW CYTOMETRY
23. Following staining, wash cells with 2mL FACS buffer and centrifuge at 400 x g, 5 min, 4 °C  and resuspend cells in >100 µL  for acquisition on a flow cytometer.