TITLE: Processing of pediatric bronchoalveolar lavage samples for single cell analysis v 3
AUTHORS: Shivanthan Shanthikumar, Liam Gubbels, 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 BRONCHOALVEOLAR LAVAGE (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 BRONCHOALVEOLAR LAVAGE (BAL)
2. For guidelines on how to safely perform bronchoscopy and lavage in children, please see:
 

SECTION: COLLECTION OF BRONCHOALVEOLAR LAVAGE (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 SINGLE CELL SUSPENSION
4. Centrifuge BAL samples at 300 x g, 7 min, 4 °C .
SECTION: PROCESSING OF BAL TO SINGLE CELL SUSPENSION
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 SINGLE CELL SUSPENSION
6. Filter cell suspension through a 70-120µm cell strainer and centrifuge filtered cell suspension 300 x g, 7 min, 4 °C.

 

SECTION: PROCESSING OF BAL TO SINGLE CELL SUSPENSION
7. Discard supernatant and resuspend cell pellet in 3 mL RPMI 2% FCS. 
SECTION: PROCESSING OF BAL TO SINGLE CELL SUSPENSION
8. 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.
SECTION: PROCESSING OF BAL TO SINGLE CELL SUSPENSION
8.1. Load 10µL of stained cells onto a Luna fluorescent counting slide and count. Record viability, total cell count, and live cell count.  

 

SECTION: PROCESSING OF BAL TO SINGLE CELL SUSPENSION
9. 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. 
SECTION: OPTION: THAWING OF CRYOPRESERVED BAL CELLS FOR SINGLE CELL ANALYSIS.
31. 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: OPTION: THAWING OF CRYOPRESERVED BAL CELLS FOR SINGLE CELL ANALYSIS.
30. 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, 7 min at room temperature. 

SECTION: OPTION: THAWING OF CRYOPRESERVED BAL CELLS FOR SINGLE CELL ANALYSIS.
29. Centrifuge the cell suspension at 300 x g, 7 min at room temperature.

SECTION: OPTION: THAWING OF CRYOPRESERVED BAL CELLS FOR SINGLE CELL ANALYSIS.
28. Rinse cryovial with 1mL warmed thaw media to recover any remaining cells and transfer to the 15mL tube.
SECTION: OPTION: THAWING OF CRYOPRESERVED BAL CELLS FOR SINGLE CELL ANALYSIS.
27. Using a pasteur pipette, transfer cells from cryovial into the 15mL tube containing warmed thaw media.
SECTION: OPTION: THAWING OF CRYOPRESERVED BAL CELLS FOR SINGLE CELL ANALYSIS.
26. Place cryovials into the water bath for cell thawing, approximately 2 min.

SECTION: OPTION: THAWING OF CRYOPRESERVED BAL CELLS FOR SINGLE CELL ANALYSIS.
25. Remove cryopreserved BAL samples from liquid nitrogen and keep on dry ice for transport to the laboratory.
SECTION: OPTION: THAWING OF CRYOPRESERVED BAL CELLS FOR SINGLE CELL ANALYSIS.
23. 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. 
SECTION: PREPARATION OF FRESH CELLS FOR FLOW CYTOMETRY
17. 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: CRYOPRESERVATION OF BAL MONONUCLEAR CELLS
10. Top up remaining cell suspension to 8mL with RPMI 2% FCS.
SECTION: CRYOPRESERVATION OF BAL MONONUCLEAR CELLS
11. Fill a 15mL tube with 2mL of Ficoll plaque plus and layer the cell suspension onto the surface of the Ficoll solution. 

 

 

SECTION: CRYOPRESERVATION OF BAL MONONUCLEAR CELLS
12. Centrifuge the layered cell suspension at 400 x g, 30 min .
SECTION: CRYOPRESERVATION OF BAL MONONUCLEAR CELLS
13. 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, 5 min, 4 °C.

 

SECTION: CRYOPRESERVATION OF BAL MONONUCLEAR CELLS
14. 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.
SECTION: CRYOPRESERVATION OF BAL MONONUCLEAR CELLS
15. Immediately place cryogenic vials into an isopropanol freezing container (e.g. Nalgene® Mr. Frosty) and transfer to -80 °C overnight.
SECTION: CRYOPRESERVATION OF BAL MONONUCLEAR CELLS
16. For long term storage, transfer vials to liquid nitrogen.
SECTION: PREPARATION OF FRESH CELLS FOR FLOW CYTOMETRY
20.  Add 25µL of antibody cocktail made up at 2X concentration and incubate for  30 min on ice .
SECTION: PREPARATION OF FRESH CELLS FOR FLOW CYTOMETRY
19. 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. 

  
 

SECTION: PREPARATION OF FRESH CELLS FOR FLOW CYTOMETRY
18. Resuspend cells in 25µL of FC-block for 5 min at Room temperature.
SECTION: PREPARATION OF FRESH CELLS FOR FLOW CYTOMETRY
21. Following staining, wash cells with 2mL FACS buffer, centrifuge at 400 x g, 5 min, 4 °C  and resuspend cells in 200µL FACS buffer for acquisition on a flow cytometer (here, a Cytek 5L Aurora). 
SECTION: PREPARATION OF FRESH CELLS FOR FLOW CYTOMETRY
22. Immediately before running the sample, filter the cell suspension through a 35 µm cell strainer ( Falcon‱ 352235).
SECTION: OPTION: THAWING OF CRYOPRESERVED BAL CELLS FOR SINGLE CELL ANALYSIS.
24. Warm thaw media (RPMI + 10% heat-inactivated FCS + 25U/mL Benzonase) to 37 °C  in a water bath.