Flow cytometric analysis to detect KIR+CD8+ T cells and KIR subtypes in human PBMCs

Antibodies and buffers

Antibodies: CD3-BUV805 (UCHT-1, BD), anti-CD56-PercP/Cy5.5 (HCD56, Biolegend), CD4-Alexa Fluor 700 (RPA-T4, Biolegend), anti-CD8-BV650 (RPA-T8, Biolegend), TCRαβ-BUV395 (IP26, BD), anti-KIR3DL1-PE (DX9, Biolegend), anti-KIR2DL2/3-PE (DX27, Biolegend), anti-KIR2DL5-PE (UP-R1, Biolegend), anti-KIR2DL1-PE (Clone 143211, R&D) and anti-KIR3DL2-PE (Clone 539304, R&D), anti-KIR3DL1-BV421 (DX9, Biolegend), anti-KIR2DL2/3-PE/Cy7 (DX27, Biolegend), anti-KIR2DL1-FITC (Clone 143211, R&D) and anti-KIR3DL2-APC (Clone 539304, R&D).

RPMI complete media: 500 ml of RPMI-1640 media supplemented with 50 ml of heat-inactivated human serum (10%), 5 ml of 100× L-Glutamax (1%) and 5 ml of penicillin/streptomycin solution (1%). 

FACS buffer: 500 ml of PBS with 2 mM EDTA and 2% heat-inactivated FBS.

Staining of PBMCs

1. Warm the RPMI complete media to 37^oC.
2. Aliquot 9 ml of pre-warmed RPMI complete media into 15 ml conical tubes and add 1 μl of Benzonase.
3. Thaw the cryovials with frozen peripheral blood mononuclear cells (PBMCs) into a 37^oC water bath. Remove the cryovial from the water bath before PBMCs are completely thawed. Do not allow the cells to warm completely to 37^oC (see Note 2).
4. Transfer 1 ml of cells from the cryovials into the 15 ml conical tubes with RPMI complete media containing Benzonase and centrifuge the cells at 600xg at 4^oC for 5 min.
5. Count the cells and determine cell viability with Trypan blue. Resuspend PBMCs at 10⁷/ml in RPMI complete media. Plate the cells in a 24-well plate.
6. Rest the PBMCs overnight (12-18h) in a 37^oC, 5% CO₂ incubator.
7. After 12-18h, transfer the PBMCs to 1.5 ml Eppendorf tubes. Centrifuge the cells at 600xg at 4^oC for 5 min. Discard the supernatants.
8. Resuspend the PBMCs in FACS buffer at <10⁷ PBMCs per 100 ul FACS buffer. Add 5 μl of Human TruStain FcX per 76.5 μl staining volume, mix and incubate at room temperature for 10 minutes.
9. Make an antibody master mix of 1 μl of anti-CD3-BUV805, 1 μl of anti-CD56-PercP/Cy5.5, 1 μl of anti-CD4-Alexa Fluor 700, 1 μl of anti-CD8-BV650, 1 μl of anti-TCRαβ-BUV395, 1 μl of anti-KIR3DL1-PE, 1 μl of anti-KIR2DL2/3-PE, 1 μl of anti-KIR2DL5-PE, 2 μl of anti-KIR2DL1-PE, 2 μl of anti-KIR3DL2-PE, 1 μl of anti-KIR3DL1-BV421, 1 μl of anti-KIR2DL2/3-PE/Cy7, 2 μl of anti-KIR2DL1-FITC, 2 μl of anti-KIR3DL2-APC and 0.5 μl of LIVE/DEAD™ Fixable Near-IR Dead Cell Stain per 100 μl staining volume. Add 18.5 μl of the antibody master mix per 81.5 μl PBMCs. Incubate at 4^oC for 30 min.
10. Wash the cell twice with 1 ml of FACS buffer. Resuspend the PBMCs at 10⁷ cells per 1 ml of FACS buffer.

Flow cytometric analysis

Cells were acquired on an LSR II flow cytometer (BD), and data were analyzed using FlowJo X. Gating strategies are shown below.