Materials and Reagents

T25 Flasks (Corning, Falcon^®, catalog numbers: 353108 )
T75 Flasks (Corning, Falcon^®, catalog numbers: 353136 )
15 ml centrifuge tubes (Corning, Falcon^®, catalog number: 352096 )
96 well plates (Corning, catalog number: 3799 )
FACS tubes
Tumor cell line (grown in flasks, in incubator at 37 °C and 5% CO₂)
RPMI 1640 (EuroClone, catalog number: ECM9106L )
Fetal bovine serum (FBS) heat-inactivated for 1 h at 56 °C (Thermo Fisher Scientific, Gibco^TM, catalog number: 10270-106 )
Penicillin/streptomycin (EuroClone, catalog number: ECB3001D )
L-glutamine (EuroClone, catalog number: ECB3000D )
EDTA (Sigma-Aldrich, catalog number: E5134 )
Phosphate buffered saline (PBS) (EuroClone, catalog number: ECB4004L )
Bovine serum albumin (BSA) (Sigma-Aldrich, catalog number: A2153-100G )
Trypan-blue
KIR-Fc fusion proteins

Recombinant Human KIR2DL1/CD158a Fc chimera protein (R&D System, catalog number: 1844-KR-050 )
Recombinant Human KIR2DL3/CD158b2 Fc chimera protein (R&D System, catalog number: 2014-KR-050 )
Recombinant Human KIR3DL1 Fc chimera protein (R&D System, catalog number: 1225-KR-050 )

Goat anti-Human Molecular Probes^TM secondary antibody, RPE conjugate (Thermo Fisher Scientific, Invitrogen, catalog number: H10104 )
Complete RPMI (see Recipes)
833 μM EDTA (see Recipes)
FACS buffer (see Recipes)

Equipment

Centrifuge, used with maximal acceleration and deceleration (Eppendorf, model: 5810R )
Incubator (5% CO₂, 37 °C) (Forma^TM Steri-Cult^TM CO₂ Incubator-Thermo Fisher Scientific)
Flow cytometer, equipped with 2 lasers (8 detectors), interfaced with PC by using DIVA Software version 6.1.3 (BD, model: FACSCanto II )

Software

DIVA software version 6.1.3
FlowJo software

Procedure

Human tumor cells are cultured in fresh RPMI medium at a cell density ≥ 0.5-0.7 x 10⁶ cells/ml in an incubator at 37 °C, 5% CO₂. For each experiment are required 0.5 x 10⁶ tumor cells.
Wash adherent cells with PBS (5 ml) and detach cells by adding 833 μM EDTA (1 ml for T25 flask and 2 ml for T75 flask) for 3-5 min in an incubator at 37 °C, 5% CO₂.
Wash cells twice with PBS (2 x 5 ml in 15 ml tube) by centrifugation at 468 x g for 7 min and re-suspend them in cold FACS buffer.
Count total viable cell number with Trypan-blue exclusion method.
Seed cells in round-bottom 96 well plates at a cell density of 1 x 10⁶ cells/well. Prepare 1 well for the control (without KIR-Fc fusion-proteins) and 1 well for each fusion protein to be tested.
Spin cells at 832 x g for 2 min at 4 °C.
Discard the supernatant and add 100 µl/well of cold FACS buffer containing single KIR-Fc fusion proteins (KIR2DL1-Fc, KIR2DL3-Fc, and KIR3DL1-Fc) diluted 1:5 and 100 μl of cold FACS buffer with none in control cells.
Incubate for 1 h at 4 °C.
Wash cells with 150 µl of cold FACS buffer and centrifuge at 832 x g at 4 °C.
Discard the supernatant and re-suspend cells in 50 µl of cold FACS buffer containing Goat anti-Human secondary antibody, RPE conjugate antibody (diluted 1:100) and mix carefully avoiding formation of bubbles.
Incubate for 25 min on ice in the dark.
Wash cells with 150 µl of cold FACS buffer and centrifuge at 832 x g at 4 °C.
Discard the supernatant and re-suspended cells in 150 µl of cold FACS buffer in FACS tubes and perform flow cytometric analysis. Viable human single cells will be selected by gating in forward scatter versus side scatter dot plot and the binding of KIR-Fc fusion proteins will be evaluated by mean fluorescence intensity (Figure 1).
Acquire data using DIVA software and analyse using FlowJo software.

Figure 1. KIR3DL1 binding to DAOY cells. Flow cytometric analysis of KIR3DL1-Fc fusion protein binding to DAOY cells (blue line). Isotype-matched negative control antibody is displayed as the red line.

Recipes

Complete RPMI
440 ml RPMI 1640
50 ml FBS
5 ml penicillin-streptomycin
5 ml L-glutamine
833 µM EDTA
Dissolve 7.3 g EDTA in 50 ml sterile water, adjust pH to 8.0
Add 83.3 µl of this solution into 50 ml sterile PBS
Filter sterilize through a 20 µm filter
Store at 4 °C
FACS buffer
50 ml PBS
1 g BSA

Acknowledgments

This protocol was adapted from Cifaldi et al. (2015). We acknowledge funding from Italian Ministry of Health (Rome, Italy) grant and the special project 5 x 1,000 Associazione Italiana per la Ricerca sul Cancro (AIRC, Milan, Italy) grant.

References

Cifaldi, L., Romania, P., Falco, M., Lorenzi, S., Meazza, R., Petrini, S., Andreani, M., Pende, D., Locatelli, F. and Fruci, D. (2015). ERAP1 regulates natural killer cell function by controlling the engagement of inhibitory receptors. Cancer Res 75(5): 824-834.
Lanier, L. L. (2005). NK cell recognition. Annu Rev Immunol 23: 225-274.
Raulet, D. H., Vance, R. E. and McMahon, C. W. (2001). Regulation of the natural killer cell receptor repertoire. Annu Rev Immunol 19: 291-330.