Processing of pediatric adenoid and tonsil samples for single cell analysis

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.

Adenoid and tonsil samples must be placed on ice and processed in the laboratory within 0h 30m 0s to 1h 0m 0s of the procedure.

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

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.

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 400x g,4°C

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.

Centrifuge the layered cell suspension at 400x g .

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 400x g,4°C

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

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.

Load 10µL of stained cells onto a LUNA fluorescent counting slide and count. Record viability, total cell count, and live cell count.

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 400x g,4°C.

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.

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 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

400x g,4°C

Resuspend cells in 25µL FACS buffer and add 15µL 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 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.pdf

Add 10µL of Brilliant Stain Buffer (Becton Dickinson) and then add 25µL of Cocktail 1A made up at 3X concentration and incubate for 0h 10m 0s

Then, directly add cockta made up at 2X concentration 1:1 with cells and incubate for 0h 30m 0s

Following staining, wash cells with 2mL FACS buffer and centrifuge at 400x g,4°C and resuspend cells in 100µL FACS buffer for acquisition on a flow cytometer (here, a Cytek 5L aurora).