TITLE: Degranulation and cytokine production (functional assay)
AUTHORS: Philippa R Kennedy

[DESCRIPTION]
Two primary functions of NK cells - their cytotoxcity and ability to produce cytokines, are measured in a flow-based assay that assesses these responses at the single cell level when NK cells are challenged with tumor cells. Cytotoxicity is not directly measured, instead it is inferred from cytotoxic granlue release. For this reason, this assay should be accompanied by a direct measure of cytotoxicity, such as aTime -lapse killing assay. Cytotoxic granlue release is measured by the accumulation of the cytotoxic granule membrane protein, CD107a (LAMP1), at the surface of the NK cell, when fusion of recycling endosomes with the lysosome is blocked by monensin. Cytokines accumulate in NK cells when Golgi transport is blocked by brefeldin A. At the end of an effector:target co-culture, these proteins and other lineage surface markers are detected by flow cytometry.

This version takes less time than the previous protocol without affecting the quality of the result. 

[BEFORE_START]
If working with frozen cells, these must be thawed and prepared the day before the assay. Book flow cytometers in advance to avoid delays in acquiring the data. Ideally, samples should be run the day after the assay was performed, but can be stored up to three days e.g. if assay was performed on a Friday.

[GUIDELINES]
The day of the assay (after effectors are plated) is long because of the 5 h incubation. Be sure to start early in the day.

During the setup of the assay, to avoid shocking the cells, pre-warm media and pipette gently, up until the staining steps. After the 5 h incubation, the aim is to stop all reactions, so pre-cool centrifuges and staining buffers to 4°C. Once cells are fixed, this cooling is no longer necessary.

The manufacturer recommend live/dead staining in PBS, but a side-by-side comparison of staining in PBS or flow buffer revealed no difference in performance of the NIR dye in our hands. 

The manufacturer recommends washing in perm buffer after staining, but a side-by-side comparison of washing in perm buffer or flow buffer revealed no difference in performance of the staining in our hands.

When working with tissue samples, blocking prior to staining is required; filtering through a 70μm filter prior to running through a flow cytometer is required; and counting beads can be beneficial for accurate assessment of cell number. 


[STEPS]
SECTION: Preparing effector and target cells
1. Effector cells (PBMCs or enriched NK cells) are resuspended in R10.
Induced pluripotent stem cell (iPSC)-derived NK cells should be resuspended in B0.
SECTION: Preparing effector and target cells
2. 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. 60 min 

SECTION: Live/dead and surface staining of cells
5. 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.
SECTION: Live/dead and surface staining of cells
5.1. The plate is spun in a centrifuge at 300g 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).

SECTION: Live/dead and surface staining of cells
5.2. The plate is spun again in the centrifuge at 300g for 5 min. The supernatant is removed and replaced with 200 μL flow buffer/well (second wash).
SECTION: Live/dead and surface staining of cells
5.3. The plate is spun for a final time at 300g for 5 min and the supernatant is removed. 
SECTION: Live/dead and surface staining of cells
7. 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 °C15 min . After the incubation, wells are topped up to 200 μL with flow buffer and washed once with flow buffer.10 min 



SECTION: Live/dead and surface staining of cells
8. Cells are resuspended in 100 μL 2% paraformaldehyde/PBS and incubated at room temperature in the dark for 10 min to fix them10 min. Afterwards, wells are topped up to 200 μL with flow buffer and washed once with flow buffer.10 min 


 

SECTION: Intracellular staining of cells
10. 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. 30 min 

SECTION: Intracellular staining of cells
11. 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. 10 min 

SECTION: Intracellular staining of cells
12. 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) 

SECTION: Intracellular staining of cells
13. 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.
SECTION: Adding inhibitors
4. 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. 240 min 

SECTION: Preparing effector and target cells
3. 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 300g for 5 min the following day in order to remove excess media.

SECTION: Intracellular staining of cells
9. Cells are washed in 150 μL perm buffer (10% Permeabilization Buffer, cat. 00-8333-56 eBioscience/distilled H20).
SECTION: Preparing effector and target cells
1.1. 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
SECTION: Live/dead and surface staining of cells
6. 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 5 min at room temperature to block non-specific staining. Proceed to surface staining without washing.