TITLE: Processing of pediatric nasal and bronchial brushing samples for single cell analysis
AUTHORS: Liam Gubbels, Shivanthan Shanthikumar, Melanie R Neeland

[DESCRIPTION]
This protocol describes the collection, processing, and cryopreservation of pediatric nasal and bronchial brushing samples for downstream single-cell analysis.

[GUIDELINES]
This is an experimental protocol for the processing of nasal and bronchial brushing samples collected from children. Sample collection must have and be compliant with Human Ethics Committee approval.

[STEPS]
SECTION: COLLECTION OF NASAL AND BRONCHIAL BRUSHINGS
1. 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.
SECTION: PROCESSING OF NASAL AND BRONCHIAL BRUSHINGS TO SINGLE CELL SUSPENSION
5. Filter cell suspension through a 70-120µm cell strainer into a new 15mL tube and centrifuge300 x g, 7 min, 4 °C .
SECTION: CRYOPRESERVATION OF NASAL AND BRONCHIAL BRUSHING CELLS
10. 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. 
SECTION: PREPARATION OF FRESH CELLS FOR FLOW CYTOMETRY
13. 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: COLLECTION OF NASAL AND BRONCHIAL BRUSHINGS
2. After obtaining informed consent from family and/or patient, collect brushings using a suitable cytology brush.

For nasal brushing:   

For bronchial brushing:        

 

SECTION: PROCESSING OF NASAL AND BRONCHIAL BRUSHINGS TO SINGLE CELL SUSPENSION
4. 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 
SECTION: PROCESSING OF NASAL AND BRONCHIAL BRUSHINGS TO SINGLE CELL SUSPENSION
6. Discard supernatant and resuspend cell pellet in 3mL RPMI 2% FCS.
SECTION: COLLECTION OF NASAL AND BRONCHIAL BRUSHINGS
3. Brushing samples must be placed on ice and processed in the laboratory within 30 min of the procedure. 
SECTION: CRYOPRESERVATION OF NASAL AND BRONCHIAL BRUSHING CELLS
11. Immediately place cryogenic vials into an isopropanol freezing container (e.g. Nalgene‱ Mr. Frosty) or Cool Cell (Corning) and transfer to -80 °C overnight.
SECTION: CRYOPRESERVATION OF NASAL AND BRONCHIAL BRUSHING CELLS
12. For long term storage, transfer the vials to liquid nitrogen.
SECTION: PREPARATION OF FRESH CELLS FOR FLOW CYTOMETRY
14. Resuspend cells in 25µL of FC-block for 5 min at Room temperature.

SECTION: PREPARATION OF FRESH CELLS FOR FLOW CYTOMETRY
15. 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
17. 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
18. Immediately before running the sample, filter the cell suspension through a 35 µm cell strainer ( Falcon‱ 352235).
SECTION: PROCESSING OF NASAL AND BRONCHIAL BRUSHINGS TO SINGLE CELL SUSPENSION
7. 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.
SECTION: PROCESSING OF NASAL AND BRONCHIAL BRUSHINGS TO SINGLE CELL SUSPENSION
7.1. 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 NASAL AND BRONCHIAL BRUSHINGS TO SINGLE CELL SUSPENSION
8. Top up remaining cell suspension to 10mL with RPMI 2% FCS, centrifuge 300 x g, 7 min, 4 °C and remove supernatant.
SECTION: PREPARATION OF FRESH CELLS FOR FLOW CYTOMETRY
16.  Add 25µL of antibody cocktail made up at 2X concentration and incubate for  30 min on ice .
SECTION: PROCESSING OF NASAL AND BRONCHIAL BRUSHINGS 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 300,000 cells prior to proceeding to cryopreservation for remaining cells.