May 08, 2024
Processing of pediatric adenoid and tonsil samples for single cell analysis
- 1Murdoch Children's Research Institute
Protocol Citation: Liam Gubbels, Shivanthan Shanthikumar, Melanie R Neeland 2024. Processing of pediatric adenoid and tonsil samples for single cell analysis. protocols.io https://dx.doi.org/10.17504/protocols.io.3byl49ewjgo5/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 20, 2024
Last Modified: May 08, 2024
Protocol Integer ID: 96965
Keywords: single-cell, flow cytometry, respiratory, Paediatric, tonsils, adenoids
Abstract
This protocol describes the collection, processing, and cryopreservation of pediatric adenoid and tonsil samples for downstream single-cell analysis.
Guidelines
This is an experimental protocol for the processing of adenoid and tonsil 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
Ficoll Paque PLUSGE HealthcareCatalog #17144003-500 ml
Cell strainer, 100 μmVWR InternationalCatalog #08-771- 19
FACS TubesContributed by users
Acridine Orange/Propidium Iodide stainLogos BiosystemsCatalog #F23991
PhotonSlideLogos BiosystemsCatalog #L12007
Human TruStain FcX™ (Fc Receptor Blocking Solution)BioLegendCatalog #422301, 422302 Petri Dish Glass 60mm x 15mm Borosilicate Pacific Laboratory ProductsCatalog #LW2387-01
Forceps (tweezers), 12.5cm, Sharp EndBio Basic Inc.Catalog #FC001.SIZE.1
Surgical scissorsContributed by users
Blades Scalpel, Handle No. 4westlabCatalog #663-930
Scalpel Blades #22westlabCatalog #663-254
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
Safety warnings
Human samples should be processed in a laboratory with appropriate biosafety infrastructure and procedures.
COLLECTION OF ADENOID AND TONSIL TISSUE
COLLECTION OF ADENOID AND TONSIL TISSUE
Prepare specimen containers for adenoid and tonsil samples by adding 10mL pre-chilled RPMI supplemented with 2% heat-inactivated fetal calf serum (referred to as RPMI 2% FCS).
After obtaining informed consent from family and/or patient, collect adenoids/tonsils at the time of clinically indicated tonsillectomy/adenoidectomy.
Note
For guidelines on how to safely collect tonsil/adenoid tissue from children, please see:
CITATION
Adenoid and tonsil samples must be placed on ice and processed in the laboratory within 00:30:00 to 01:00:00 of the procedure.
PROCESSING OF ADENOID AND TONSIL TISSUE TO CELL SUSPENSION
PROCESSING OF ADENOID AND TONSIL TISSUE TO CELL SUSPENSION
1h 50m
Place the tissue in a glass cell culture plate with 10mL RPMI 2% FCS. Remove any visible blood clots, fat, and connective tissues with forceps and scissors/scalpel
10m
Transfer the trimmed adenoid/tonsil tissue to a new glass cell culture plate containing 10mL RPMI 2% FCS. Mince the tissue into a fine paste using scissors or a scalpel.
10m
Muddle the tissue using a plunger from a sterile syringe to dissociate the cells from the tissue, then filter the cell suspension through a 100µm cell strainer into a 50mL tube. Centrifuge the cell suspension 400 x g, 4°C, 00:05:00
Note
Cell isolation can also be done using a gentleMACS tissue dissociator
15m
Remove the supernatant and resuspend the cell pellet in 8mL RPMI 2% FCS. Fill a 15mL tube with 2mL of Ficoll plaque plus and layer the adenoid/tonsil cell suspension onto the surface of the Ficoll solution.
Note
Layer the cell suspension slowly to prevent the Ficoll solution from mixing with the cells.
5m
Centrifuge the layered cell suspension at 400 x g, Room temperature, 00:30:00 , 4 Acceleration and NO brake .
45m
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, 4°C, 00:05:00
Note
When collecting the cells, try to avoid Ficoll solution as much as possible.
5m
Discard supernatant and resuspend cell pellet in 3 mL RPMI 2% FCS.
5m
Prepare cell suspension for cell counting. Here, we use AO/PI and the LUNA FL counter. Dilute cell suspension in a microcentrifuge tube in RPMI 2% FCS at a ratio of 1:10 for adenoids and 1:100 for tonsils. Remove 18µL of diluted cells and place into a new microcentrifuge tube for cell counting. Add 2 µL of AO/PI to the count tube and mix well.
5m
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 also be performed manually using a haemocytometer, or using other automated cell counters.
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 500,000 cells.
Top up the cell suspension to 10mL with RPMI 2% FCS and centrifuge 400 x g, 4°C, 00:05:00 .
5m
CRYOPRESERVATION OF ADENOID AND TONSIL MONONUCLEAR CELLS
CRYOPRESERVATION OF ADENOID AND TONSIL MONONUCLEAR CELLS
10m
Discard supernatant and 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 1-20 million cells/mL. Transfer cells to cryogenic vial.
10m
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 CELLS FOR FLOW CYTOMETRY
PREPARATION OF CELLS FOR FLOW CYTOMETRY
5m
Resuspend cell suspension for fixable viability staining according to manufacturers' instructions (e.g. the LIVE/DEAD‱ Fixable UV Blue Stain from Invitrogen/ThermoFisher).
Following the required incubation, stop the reaction by the addition of 1mL staining buffer (2% heat-inactivated FCS in PBMS 2 mM EDTA, herein referred to as FACS buffer) and centrifuge at
400 x g, 4°C, 00:05:00
5m
Resuspend cells in 25µL FACS buffer and add 15µL 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 31-plex spectral cytometry panel that we routinely use on cells isolated from tonsil and adenoid tissue. All of the following steps are related to this panel.
tonsil_adenoid_blood_panel.pdf127KB Add 10µL of Brilliant Stain Buffer (Becton Dickinson) and then add 25µL of Cocktail 1A made up at 3X concentration and incubate for 00:10:00 In the dark at room temperature
10m
Then, directly add cockta made up at 2X concentration 1:1 with cells and incubate for 00:30:00 In the dark at room temperature
30m
Following staining, wash cells with 2mL FACS buffer and centrifuge at 400 x g, 4°C, 00:05:00 and resuspend cells in 100µL FACS buffer for acquisition on a flow cytometer (here, a Cytek 5L aurora).
Note
Panels 1A and 1B were adapted from: "OMIP-069: Forty-Color Full Spectrum Flow Cytometry Panel for Deep Immunophenotyping of Major Cell Subsets in Human Peripheral Blood" and we thank the authors for their detailed methods.
CITATION
5m