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

[DESCRIPTION]
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.

[STEPS]
SECTION: COLLECTION OF ADENOID AND TONSIL TISSUE
1. 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).
SECTION: COLLECTION OF ADENOID AND TONSIL TISSUE
2. After obtaining informed consent from family and/or patient, collect adenoids/tonsils at the time of clinically indicated tonsillectomy/adenoidectomy.

 

SECTION: COLLECTION OF ADENOID AND TONSIL TISSUE
3. Adenoid and tonsil samples must be placed on ice and processed in the laboratory within 30 min to 60 min of the procedure.

SECTION: PROCESSING OF ADENOID AND TONSIL TISSUE TO CELL SUSPENSION
4. 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
SECTION: PROCESSING OF ADENOID AND TONSIL TISSUE TO CELL SUSPENSION
5. 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. 
SECTION: PROCESSING OF ADENOID AND TONSIL TISSUE TO CELL SUSPENSION
6. 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, 5 min, 4 °C 

 

SECTION: PROCESSING OF ADENOID AND TONSIL TISSUE TO CELL SUSPENSION
7. 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. 

 

SECTION: PROCESSING OF ADENOID AND TONSIL TISSUE TO CELL SUSPENSION
8. Centrifuge the layered cell suspension at 400 x g, 30 min .
SECTION: PROCESSING OF ADENOID AND TONSIL TISSUE TO CELL SUSPENSION
9. 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, 5 min, 4 °C 

 

SECTION: PROCESSING OF ADENOID AND TONSIL TISSUE TO CELL SUSPENSION
10. Discard supernatant and resuspend cell pellet in 3 mL RPMI 2% FCS. 
SECTION: PROCESSING OF ADENOID AND TONSIL TISSUE TO CELL SUSPENSION
11. 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.
SECTION: CRYOPRESERVATION OF ADENOID AND TONSIL MONONUCLEAR CELLS
14. 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.
SECTION: CRYOPRESERVATION OF ADENOID AND TONSIL MONONUCLEAR CELLS
15. 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 ADENOID AND TONSIL MONONUCLEAR CELLS
16. For long term storage, transfer the vials to liquid nitrogen.
SECTION: PREPARATION OF CELLS FOR FLOW CYTOMETRY
17. Resuspend cell suspension for fixable viability staining according to manufacturers' instructions (e.g. the LIVE/DEAD‱ Fixable UV Blue Stain from Invitrogen/ThermoFisher). 
SECTION: PREPARATION OF CELLS FOR FLOW CYTOMETRY
18. 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, 5 min, 4 °C 
SECTION: PROCESSING OF ADENOID AND TONSIL TISSUE TO CELL SUSPENSION
11.1. 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 ADENOID AND TONSIL TISSUE TO CELL SUSPENSION
12. 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. 
SECTION: PREPARATION OF CELLS FOR FLOW CYTOMETRY
19. Resuspend cells in 25µL FACS buffer and add 15µL FC-block for 5 min at Room temperature
SECTION: PREPARATION OF CELLS FOR FLOW CYTOMETRY
20. 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.

  
SECTION: PREPARATION OF CELLS FOR FLOW CYTOMETRY
22. Then, directly add cockta made up at 2X concentration 1:1 with cells and incubate for 30 min 
SECTION: PREPARATION OF CELLS FOR FLOW CYTOMETRY
23. Following staining, wash cells with 2mL FACS buffer and centrifuge at 400 x g, 5 min, 4 °C  and resuspend cells in 100µL FACS buffer for acquisition on a flow cytometer (here, a Cytek 5L aurora). 

 

SECTION: PROCESSING OF ADENOID AND TONSIL TISSUE TO CELL SUSPENSION
13. Top up the cell suspension to 10mL with RPMI 2% FCS and centrifuge 400 x g, 5 min, 4 °C.
SECTION: PREPARATION OF CELLS FOR FLOW CYTOMETRY
21. Add 10µL of Brilliant Stain Buffer (Becton Dickinson) and then add 25µL of Cocktail 1A made up at 3X concentration and incubate for 10 min