Processing of pediatric nasal and bronchial brushing samples for single cell analysis

Prepare collection tubes for nasal and bronchial brushing samples by adding 5mL of pre-chilled RPMI supplemented with 2% heat-inactivated fetal calf serum (referred to as RPMI 2% FCS) to a 15mL tube labelled with the study/patient ID.

After obtaining informed consent from family and/or patient, collect brushings using a suitable cytology brush.

For nasal brushing:

For bronchial brushing:

Brushing samples must be placed on ice and processed in the laboratory within 0h 30m 0s of the procedure.

Repeatedly pipette media onto brushing to dislodge cells. Do this for at least 1 minute

per brushing. Remove cytology brush and top up the cell suspension to 10mL with RPMI 2% FCS

Filter cell suspension through a 70-120µm cell strainer into a new 15mL tube and centrifuge300x g,4°C .

Discard supernatant and resuspend cell pellet in 3mL RPMI 2% FCS.

Prepare cell suspension for cell counting. Here, we use AO/PI and the LUNA FL counter. Remove 18µL 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.

Top up remaining cell suspension to 10mL with RPMI 2% FCS, centrifuge 300x g,4°C and remove supernatant.

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 300,000 cells prior to proceeding to cryopreservation for remaining cells.

Resuspend cells at a ratio of 1:1 in 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) or Cool Cell (Corning) and transfer to -80°C overnight.

For long term storage, transfer the vials to liquid nitrogen.

Resuspend cell suspension for fixable viability staining according to manufacturers' instructions (e.g. the LIVE/DEAD^TM 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 25µL of FC-block for 0h 5m 0s at Room temperature.

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.pdf

Add 25µL of antibody cocktail made up at 2X concentration and incubate for 0h 30m 0s On ice .

Following staining, wash cells with 2mL FACS buffer, centrifuge at 400x g,4°C and resuspend cells in 200µL FACS buffer for acquisition on a flow cytometer (here, a Cytek 5L Aurora).

Immediately before running the sample, filter the cell suspension through a 35 µm cell strainer ( Falcon^TM 352235).