Cd56 pecy7 b159

Natural killer cell percentage and phenotype were evaluated in PBMC obtained from AML patients and HDs. The following anti-human mAbs were used for flow cytometry: CD56-FITC (NCAM16.2), CD56-PE (MY31), CD14-PE (MØP9), CD3-PerCP (SK7) all from BD Biosciences (San Jose, CA, USA); CD56-PECy7 (B159), CD16-APC Cy7 (3G8), NKG2D-PE (1D11), CD226-PE (DX11), CD107a-FITC (H4A3), CD107b-FITC (H4B4), CD86-FITC (2331(FUN-1)), CD1a-FITC (HI149) all from BD Pharmingen (San Diego, CA, USA); and NKp30-PE (AF29-4D12), NKp30-APC (AF29-4D12), NKp46-PE (9E2), NKp46-APC (9E2), CD3-VioBlue (BW264/56) all from Miltenyi Biotec (Bergisch Gladbach, Germany). Prior to use, mAbs were titrated to establish optimal staining dilutions. Isotype-matched immunoglobulins were included in all experiments as negative controls. Mean relative fluorescence intensity was calculated by dividing the mean fluorescent intensity (MFI) of the relevant mAb by the MFI of its isotype control.

Cell surface staining of study PBMCs was performed using fluorophore-conjugated antibodies against the following proteins: CD45-V450 (HI30, BD Biosciences), CD3-V500 (SP34-32, BD Biosciences), TCR γδ-PE (B1, BD Biosciences), CD8-FITC (HIT8a, BD Biosciences), CD4-APC H7 (SK3, BD Biosciences), CD56-PE Cy7 (B159, BD Biosciences), HLA-DR-APC (TU36, BD Biosciences), CD127-APC R700 (HIL-7R-M21, BD Biosciences), CD25-PE CF594 (M-A521, BD Biosciences) CD38-Qdot655 (HIT2, Thermo Fisher Scientific). Briefly, Cells were washed with PBS, incubated with human TruStain FcX (Biolegend) to block Fc receptors, and then incubated with antibodies diluted in stain buffer (BD Pharmigen) for 2 hours at 25°C. After staining cells were washed twice with PBS and fixed with 2% paraformaldehyde. Following staining, events were collected using a LSR Fortessa flow cytometer (BD Biosciences) and fluorophore compensation was calculated using single stained PBMC controls on the BD FACSDiva software package. Resulting Data were analyzed using FlowJo Software (Tree Star) and GraphPad Prism (GraphPad Software). Statistically significant differences in PBMC cell population percentages between iRBC lysate and uRBC co-cultures for each patient group (symptomatic vs. asymptomatic) were identified using a two-tailed Wilcoxon matched pairs signed rank test. P-values of < 0.05 were considered statistically significant.

Fresh tumor tissues were washed with PBS, cut into small pieces, and digested with collagenase type IV (1 mg/mL, Sigma) and DNase I (10 μg/mL, Sigma) in the RPMI 1640 medium for 30 min at 37 °C. Tumor suspensions were passed through a 70 μm filter. Cell suspensions and tumor suspensions were surface-labeled for antibodies for 30 min at 4 °C. For intracellular staining, cells were fixed and permeabilized using a Transcription Factor Buffer Set according to the manufacturer’s instructions (#562574, BD Biosciences) and incubated with antibodies for 30 min at 4 °C. The stained cells were analyzed on an LSRFortessa X-20 (BD Biosciences). The specific antibodies were used for flow cytometry as follows: Anti-Human CD8-FITC (SK1, #344704, Biolegend), CD56-PE/Cy7 (B159, #557747, BD Biosciences), Granzyme B-Brilliant Violet 421 (QA18A28, #396414, Biolegend), IFN-γ-APC (4S.B3, #502512, Biolegend), CD3-PerCP/Cyanine5.5 (UCHT1, #300430, Biolegend), CD45-BV510 (HI30, #563204, BD Biosciences), TNF-α-PE (MAb11, #559321, BD Biosciences), HLA-F-APC (3D11, #373208, Biolegend), and Fixable Viability Stain 780 (#565388, BD Biosciences).