Cd56 apc

Immunophenotypic analysis was performed using three colour flow cytometry on a FACS Calibur (Becton Dickinson) and six colour flow cytometry on a FACS Canto (Becton Dickinson). Antibodies used were CD45 FITC, 2DL2/3 FITC, CD56 PE, NKG2D PE, CD3 PerCP, Streptavidin PerCP, CD56 PE-Cy7, 2DL1 APC, CD56 APC, CD3 APC-Cy7, 3DL1 biotin (all Becton Dickinson). Ig isotype controls were used where appropriate. Lymphocyte gating was performed using the forward scatter versus side scatter plot and 10 000 events were acquired. NK cells were identified as CD3⁻CD56⁺, T cells as CD3⁺CD56⁻, and CD56⁺ T cells as CD3⁺CD56⁺. Data was analysed using Cell Quest/FACS Diva software.

Samples were delivered to the laboratory de-identified using a unique code by an independent blood collector. Eighty ml of blood was used for peripheral blood mononuclear cells (PBMC) isolation by density gradient centrifugation using Ficoll (GE Healthcare, Uppsala, Sweden) as previously described [45 ]. Isolated PBMCs were adjusted to a final concentration of 5 × 10⁷ cells/ml for NK cell isolation.
NK cells were isolated by immunomagnetic selection using the EasySep Negative Human NK cell Enrichment Kit (Stem Cell Technologies, Vancouver, BC, Canada). NK cell purity was defined by CD3⁻CD56⁺ surface expression using flow cytometry (Additional file 1: Fig. S1). Specifically, NK cells were incubated for 20 min at room temperature in the presence of CD3 PE-Cy7 (5 µl/test) and CD56 APC (20 µl/test) monoclonal antibodies (Becton Dickinson [BD] Biosciences, San Jose, CA, USA). Cells were acquired at 10,000 events using the Accuri C6 flow cytometer (BD Biosciences, San Diego, CA, USA). Acceptable NK cell purity was ≥ 90% (Additional file 1: Fig. S2).

Samples were deidentified using a unique code and delivered to the laboratory. Eighty ml of blood was used for peripheral blood mononuclear cells (PBMC) isolation by density gradient centrifugation using Ficoll (GE Healthcare, Uppsala, Sweden) as previously described [34 (link)]. PBMCs were stained with trypan blue (Invitrogen, Carlsbad, CA, USA) to determine cell count and cell viability. PBMCs were adjusted to a final concentration of 5 × 10⁷ cells/ml for NK cell isolation.
NK cells were isolated by immunomagnetic selection using the EasySep Negative Human NK cell Enrichment Kit (Stem Cell Technologies, Vancouver, BC, Canada). Approximately 2.5–4 × 10⁶ cells NK cells were isolated and used for Ca²⁺ imaging experiments. NK cell purity was defined by CD3⁻CD56⁺ surface expression using flow cytometry (Additional file 1: Figure S1). Specifically, NK cells were incubated for 20 min at room temperature in the presence of CD3 PE-Cy7 (5 µl/test) and CD56 APC (20 µl/test) monoclonal antibodies (Becton Dickinson [BD] Biosciences, San Jose, CA, USA). Cells were acquired at 10,000 events using the Accuri C6 flow cytometer (BD Biosciences, San Diego, CA, USA). The average NK cell purity (%) for this study was 86.73 ± 9.71 (Additional file 1: Figure S2).

Peripheral Blood was collected in 5 ml EDTA tubes at cycles: 1, 2, 3, 6, 9, and 12. Blood samples were taken at day 1 of each cycle before the administration of IL2 and at day 8. The phenotype of T-cells was evaluated with the following monoclonal antibodies: CD57-FITC, PE‐γδ TCR, CD3 Per-Cp, CD45RA PE-Cy7, CD45RO, CD45APC-Cy7, CD27 V500, CD25 PE-Cy7, CD4 FITC, CD69 Per-Cp, CD3 V500, CD127 PE, CD56 APC, CD8 V500 (Becton Dickinson, San Jose, CA, USA). Corresponding irrelevant isotype‐matched mouse monoclonal antibodies were used as negative controls. Briefly, 100 μl of peripheral blood are incubated for 20 min with the respective monoclonal antibodies (7 μl for FITC and PE, 5 μl for the other fluorochromes). Then 2 ml of lysant (NH4Cl) were added for erythrocyte lysis and then centrifuged at 2.1 rpm for 5’. The resulting pellet, after removal of the supernatant, was re-suspended with 1 ml of PBS. Finally, 30 × 10⁴ total events or 10 × 10⁴ events in the lymphocyte gate were acquired on a FacsCanto II, equipped with three lasers (violet 405 nm, blue 488 nm, and red 633 nm) and analyzed with FacsDiva software (BD Bioscience, Erembodegem, Belgium).