Processing of pediatric bronchoalveolar lavage samples for single cell analysis

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:

BAL samples must be placed on ice and processed in the laboratory within 30 minutes -1 hour of the procedure.

Centrifuge BAL samples at 300x g,4°C .

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).

Filter cell suspension through a 70-120µm cell strainer and centrifuge filtered cell suspension 300x g,4°C.

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 300x g,4°C while performing cell count.

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:

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.

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.

Warm thaw media (RPMI + 10% heat-inactivated FCS + 25U/mL Benzonase) to 37°C in a water bath.

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 0h 2m 0s.

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 300x g at room temperature.

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 300x g at room temperature.

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.

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 400x g,4°C.

Resuspend cells in human FC-block according to manufacturers’ instructions for 0h 5m 0s at room temperature.

Add required antibody cocktail made up at 2X concentration 1:1 with the cells and incubate for 0h 30m 0s on ice. For examples of relevant antibody stains for pediatric lung samples, please see our published work below.

Following staining, wash cells with 2mL FACS buffer and centrifuge at 400x g,4°C and resuspend cells in >100µL for acquisition on a flow cytometer.