Degranulation and cytokine production (functional assay)

Effector cells (PBMCs or enriched NK cells) are resuspended in R10 .

Induced pluripotent stem cell (iPSC)-derived NK cells should be resuspended in B0 .

R10 media recipe:

• RPMI cat. 2240-089, Gibco
• 10% heat inactivated fetal calf serum cat. 26140079, Gibco
• 100 U/ml Penicillin & 100 μg/ml Streptomycin cat. 15140122, Gibco

B0 media recipe:

• 60% DMEM cat. 10-017-CV Corning
• 30% Hams F12 cat. 10-080-CV Corning
• 10% heat inactivated AB serum cat. HP1022 Valley Biomedical Inc
• 100 U/ml Penicillin & 100 μg/ml Streptomycin cat. 15140122, Gibco
• 20 μM β-mercaptoethanol cat. M7522, Sigma Aldrich
• 50 μM Ethanolamine cat. E0135, Sigma Aldrich
• 20 μg/ml Ascorbic Acid cat. A4544, Sigma Aldrich
• 5 ng/ml Sodium Selenite (Na2SeO3) cat. S5261, Sigma Aldrich

Target cells are added to each well at an effector:target ratio of 2:1. At the same time, FITC conjugated anti-CD107a (clone H4A3, BioLegend; batch specific, but normally 3 μL/well ) is added to each well. The cells are then incubated at 37°C 5% CO₂. 1h 0m 0s

5 x 10⁵ cells/well are added to a U-bottomed 96 well plate.

These numbers can be decreased to 1 x 10⁵ effector cells/well if the number of effector cells is limited, so long as target numbers are decreased accordingly.

If thawing effector cells, these should be plated one day in advance in 200 μL R10 (PBMC) or 200 μL B0 with 50 U/mL IL-2 (iPSC-NK cells) and cultured at 37°C 5% CO₂ overnight . This allows recovery of surface proteins and functionality. In this case, plates are spun at 300 g for 5 min the following day in order to remove excess media.

One hour after the addition of anti-CD107a, cells are given monensin (GolgiStop Cat. No. 554724, BD Biosciences) and brefeldin A (GolgiPlug Cat. No. 555029, BD Biosciences). GolgiStop (1/150) and GolgiPlug (1/100) are diluted in R10 and 20 μL is added to each well. Cells are incubated for a further 4 h at 37°C 5% CO₂. 4h 0m 0s

Cells are washed twice, as defined below, in flow buffer (1% AB serum, 0.5mM EDTA in PBS) . This definition also applies to subsequent washes.

The plate is spun in a centrifuge at 300 g for 5 min (this is approximately 1200rpm in a large benchtop centrifuge) . The supernatant is removed and replaced with 200 μL flow buffer/well (first wash).

The plate is spun again in the centrifuge at 300 g for 5 min. The supernatant is removed and replaced with 200 μL flow buffer/well (second wash).

The plate is spun for a final time at 300 g for 5 min and the supernatant is removed.

Optional: If working with processed tissues, prepare 5μL Fc Receptor Blocking Solution (cat. 422302, BioLegend) in 100μL flow buffer. Resuspend each well in 20μL of this blocking solution and incubate for 5 min at room temperature to block non-specific staining. Proceed to surface staining without washing. 0h 5m 0s at room temperature to block non-specific staining. Proceed to surface staining without washing.

Cells are resuspended in 50 μL flow buffer containing:

1. anti-CD56-PE-Cy7 (clone HCD56, Biolegend; batch specific, but normally 5 μL/test )

2. anti-CD3-PECF594 (clone UCHT1, BD Horizon, batch specific, but normally 1 μL/test )

3. Live/Dead Fixable Near-IR Staining Kit, cat. L-34976, Thermo Fisher ( 1/1000 dilution )

Incubate for 15 min at 4 °C0h 15m 0s . After the incubation, wells are topped up to 200 μL with flow buffer and washed once with flow buffer.0h 10m 0s

Cells are resuspended in 100 μL 2% paraformaldehyde/PBS and incubated at room temperature in the dark for 10 min to fix them0h 10m 0s. Afterwards, wells are topped up to 200 μL with flow buffer and washed once with flow buffer.0h 10m 0s

Cells are washed in 150 μL perm buffer (10% Permeabilization Buffer, cat. 00-8333-56 eBioscience/distilled H20) ₂0).

Permeabilized cells are resuspended in 50 μL perm buffer containing:

1. BV650 conjugated IFNγ (4S.B3, BioLegend; batch specific, but normally 3 μL/test )

2. BV421 conjugated TNFa (Mab11, Biolegend; batch specific, but normally 5 μL/test ).

They are incubated for 30 min at 4 °C in the dark. 0h 30m 0s

After staining, wells are topped up to 200 μL with flow buffer and washed once with flow buffer. Cells are resuspended in 200 μL of flow buffer and transferred to bullet tubes. 0h 10m 0s

The tubes are covered and stored in the dark at 4 °C until they are ready to be run on a flow cytometer (LSR II, BD Biosciences).

To be able to compare NK cell numbers or tumor cell numbers between samples, run the flow cytometer at a consistent speed (e.g. high) and acquire data for a set time (e.g. 60s)

Data is analyzed using FlowJo software (Tree Star Inc., RRID:SCR_008520)

Degranulation (CD107a+) and cytokine production (IFNγ+ or TNFα+) are assessed for the live (dead cell marker-) NK cell (CD56+ CD3-) population.