Redirected lysis (P815 functional assay)

Optional: Label specific cell subsets prior to this assay with Cell Trace Violet (CTV) according to the manufacturer's instructions to examine the behavior of a particular cell subset.

Option 1: Label effector cells (peripheral blood mononuclear cells 'PBMC' or NK cells) with CTV before combining them with unlabeled subsets (e.g. monocytes) to see how the effector cells perform in combination. Care must be taken when determining effector: target cell ratios.

Option 2: Label effector cells with CTV to combine this assay with a proliferation assay. Perform this functional assay at the end of the proliferation assay.

Option 3: Label P815 cells with CTV to combine this assay with a direct measure of target cell death. At the end of the assay, when analyzing samples on a flow cytometer, count the relative number of live P815 for each treatment condition by running the flow cytometer at a constant flow rate and analyzing the number of live CTV+ cells obtained within a specified time limit e.g. 60s.

P815 cells are counted and resuspended at 2.5x10⁶ cells/mL in R10 (RPMI + 10% fetal bovine serum + 1% penicillin streptomycin). 100 μl (2.5x10⁵ cells) per well are added to a 96 well U-bottom plate. Anti-human receptor mAb (e.g. anti-CD16, anti-NKp30, anti-NKG2D, anti-DNAM1, anti-CD2, mIgG1) at 1 μg/ml are added to individual wells containing P815 and to the same number of wells containing R10 alone. The plate is incubated for 30 minutes at room temperature.

During the incubation, effector cell populations are counted and resuspended in R10. At the end of the incubation, 5x10⁵ effector cells and anti-CD107a-FITC antibody (5 μl/well) are added to each well, into a final volume of 200 μl. The plate is incubated at 37°C 5% CO₂ for 1 h.

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.

Cells are washed twice in PBS, as defined below. This definition also applies to subsequent washes.

The plate is spun in a centrifuge at 300 g for 5 min. The supernatant is removed and replaced with 200 μL PBS/well (first wash).

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

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

Cells are resuspended in 200 μL PBS with a dead cell marker (1/1000 dilution; Live/Dead Fixable Aqua Staining Kit, Cat. No: L-34966, Thermo Fisher) and incubated for 30 min at 4°C in the dark.

Cells are washed twice in flow buffer (1% AB serum, 0.5mM EDTA in PBS).

After the final spin, wells are resuspended in 50 μL of flow buffer containing anti-CD56-PE-Cy7 (2 μl / well) and anti-CD3-PECF594 (1 μL / well). The plate is incubated at 4°C for 15 min in the dark. After the incubation, wells are topped up to 200 μL with flow buffer and washed once with flow buffer.

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

Cells are resuspended in 100 μL 0.1% Triton X/PBS and incubated at room temperature in the dark for 5 min to permeabilize them. Afterwards, wells are topped up to 200 μL with flow buffer and washed once with flow buffer.

Permeabilized cells are resuspend in 100 μL of flow buffer containing anti-IFNγ-BV650 (3 μL / well) and anti-TNFα-AF647 (5 μL / well). The plate is incubated for 30 min at 4°C in the dark.

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.

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).

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 and subdivided according to cell trace labeling.