May 08, 2024
Processing of pediatric nasal and bronchial brushing samples for single cell analysis
- 1Murdoch Children's Research Institute
Protocol Citation: Liam Gubbels, Shivanthan Shanthikumar, Melanie R Neeland 2024. Processing of pediatric nasal and bronchial brushing samples for single cell analysis. protocols.io https://dx.doi.org/10.17504/protocols.io.5jyl8p379g2w/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: March 13, 2024
Last Modified: May 08, 2024
Protocol Integer ID: 96676
Keywords: single-cell, flow cytometry, respiratory, Paediatric, nasal brushing, bronchial brushing
Funders Acknowledgement:
Chan Zuckerberg Initiative
Grant ID: 2021-237883
Abstract
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.
Materials
RPMI-1640Sigma AldrichCatalog #R5886
Fetal Bovine Serum
1X PBS (Phosphate-buffered saline )
DMSO (dimethyl sulfoxide)Sigma AldrichCatalog #D8418
conical tubes, 50ml
conical tubes, 15ml
1.5 mL Eppendorf tubesContributed by users
Corning® cell strainerCorningCatalog #CLS431751-50EA
FACS TubesContributed by users
Acridine Orange/Propidium Iodide stainLogos BiosystemsCatalog #F23991
PhotonSlideLogos BiosystemsCatalog #L12007
Human TruStain FcX™ (Fc Receptor Blocking Solution)BioLegendCatalog #422301, 422302
Cytology Brush Ring Handle 3.00mm X 120.00cmConmedCatalog #149
Cytology Brush Single-use Respiratory Compatible Channel - 1.2mm , 2mm brush width 6mm lengthOlympusCatalog #BC-203D-2006
cryovialContributed by users
Flow antibodies: 
Flow cytometry panels.pdf301KB
Flow cytometry panels.pdf301KB Equipment
Aurora - 5L Configuration
NAME
Spectral Flow Cytometer
TYPE
Cytek Aurora
BRAND
N/A
SKU
5L Configuration
SPECIFICATIONS
Protocol materials
Fetal Bovine Serum
Materials
Human TruStain FcX™ (Fc Receptor Blocking Solution)BioLegendCatalog #422301, 422302
Materials
Acridine Orange/Propidium Iodide stainLogos BiosystemsCatalog #F23991
Materials
Cytology Brush Ring Handle 3.00mm X 120.00cmConmedCatalog #149
Materials, Step 2
Corning® cell strainerCorningCatalog #CLS431751-50EA
Materials
PhotonSlideLogos BiosystemsCatalog #L12007
Materials
DMSO (dimethyl sulfoxide)Merck MilliporeSigma (Sigma-Aldrich)Catalog #D8418
Materials
Cytology Brush Single-use Respiratory Compatible Channel - 1.2mm , 2mm brush width 6mm lengthOlympusCatalog #BC-203D-2006
Materials, Step 2
RPMI-1640Merck MilliporeSigma (Sigma-Aldrich)Catalog #R5886
Materials
conical tubes, 15ml
Materials
FACS Tubes
Materials
cryovial
Materials
conical tubes, 50ml
Materials
1.5 mL Eppendorf tubes
Materials
Safety warnings
Human samples should be processed in a laboratory with appropriate biosafety infrastructure and procedures
COLLECTION OF NASAL AND BRONCHIAL BRUSHINGS
COLLECTION OF NASAL AND BRONCHIAL BRUSHINGS
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: Cytology Brush Ring Handle 3.00mm X 120.00cmConmedCatalog #149
For bronchial brushing: Cytology Brush Single-use Respiratory Compatible Channel - 1.2mm , 2mm brush width 6mm lengthOlympusCatalog #BC-203D-2006
Note
For guidelines on how to safely perform bronchial/nasal brushings in children, please see:
CITATION
CITATION
LINK
https://doi.org/Brushing samples must be placed on ice and processed in the laboratory within 00:30:00 of the procedure.
30m
PROCESSING OF NASAL AND BRONCHIAL BRUSHINGS TO SINGLE CELL SUSPENSION
PROCESSING OF NASAL AND BRONCHIAL BRUSHINGS TO SINGLE CELL SUSPENSION
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 centrifuge300 x g, 4°C, 00:07:00 .
7m
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.
Note
Cell counting can be performed manually using a haemocytometer, or using other automated cell counters.
Top up remaining cell suspension to 10mL with RPMI 2% FCS, centrifuge 300 x g, 4°C, 00:07:00 and remove supernatant.
7m
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.
CRYOPRESERVATION OF NASAL AND BRONCHIAL BRUSHING CELLS
CRYOPRESERVATION OF NASAL AND BRONCHIAL BRUSHING 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.
PREPARATION OF FRESH CELLS FOR FLOW CYTOMETRY
PREPARATION OF FRESH CELLS FOR FLOW CYTOMETRY
45m
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, 4°C, 00:05:00
5m
Resuspend cells in 25µL of FC-block for 00:05:00 at Room temperature .
5m
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.pdf114KB CITATION
Add 25µL of antibody cocktail made up at 2X concentration and incubate for 00:30:00 On ice .
30m
Following staining, wash cells with 2mL FACS buffer, centrifuge at 400 x g, 4°C, 00:05:00 and resuspend cells in 200µL FACS buffer for acquisition on a flow cytometer (here, a Cytek 5L Aurora).
5m
Immediately before running the sample, filter the cell suspension through a 35 µm cell strainer ( Falcon‱ 352235).