Anti cd56 af700

Cell surface staining was performed on NK cells after 18h of incubation with Control-CM, ALG-CM, or 3mM CaCl₂ in R10 complete medium. Cells were stained in staining buffer with fluorochrome-linked monoclonal antibodies: anti-CD14-FITC (BD Biosciences), anti-CD3-FITC (Biolegend), anti-CD56-AF700 (BD Biosciences), anti-CD16-BUV395 (BD Biosciences), anti-CD69-BUV737 (BD Biosciences), anti-DNAM-1-PE-Vio770 (Miltenyi), anti-NKp30- anti-BV421 (BD Biosciences), anti-NKp46-BV786 (BD Biosciences), anti-NKG2D-BV650 (BD Biosciences), anti-KIR2D-PE (Miltenyi), anti-KLRG1-APC-Vio770 (Miltenyi), anti-CD95-APC (Biolegend), anti-CD85j-PE-Cy5 (BD Biosciences), anti-CD158e1/e2 PerCP-Vio700 (Miltenyi), anti-TIGIT-BV605 (Biolegend), and Fixable Viability Dye eFluor 506. The expression of activating or inhibitory receptors was analyzed on NK cell subsets based on CD16 and/or CD56 expression. Samples were analyzed using an LSRFortessa cytometer (BD Biosciences), and results were generated by FlowJo software.

NK cells were isolated from healthy donors by negative selection using magnetic‐activated cell sorting (Miltenyi Biotec Bergisch). Isolated NK cells were maintained overnight in RPMI-1640 (Lonza, Basel, Switzerland) supplemented with 5% human serum (C.C pro, Oberdorla, Germany) and IL‐2 (100U/mL) (Prepotech, New Jersey, USA). HLA-silenced and non-silenced LSCs unstimulated or IFNɣ-stimulated (100ng/mL) for 48 hours were used to evaluate NK cell degranulation. At the day of the experiment, LSCs were cultured with NK cells in a target: effector ratio of 1:2 in 96-well plates (Falcon, Corning Brand) for 5 hours at 37°C. After incubation time, NK cells were collected, stained with anti- CD3-APC/Cy7 (clone: HIT3a; Biolegend, California, USA), anti-CD56-AF700 (clone: B159; BD Biosciences, New Jersey, USA) and CD107a-PE (clone: H4A3; Biolegend, California, USA) and analyzed by flow cytometry. NK cell activation was evaluated by measuring CD107a expression as a marker of degranulation.

Quality control of LP, preDS, and DS was performed as previously described (Tischer et al., 2014b (link)). Total CD45⁺ leukocytes, viability, and frequencies as well as total cell numbers were determined using Trucount Absolute Counting Tubes (BD Biosciences) and the following staining reagents: anti-CD45 APC-H7 and anti-CD3 FITC mAbs and 7-AAD (all BD Biosciences). After staining, erythrocytes were lysed using Lysing Solution (Beckman Coulter) and samples were acquired on a BD FACSCanto 10c with at least 10,000–50,000 events in the Trucount beads gate. Purity, memory phenotype and cellular composition were analyzed using combinations of the following staining reagents: anti-CD45 APC-H7, anti-CD3 FITC, anti-CD4 AF700, anti-CD8 APC, anti-CD14 BV510, anti-CD19 BV510, anti-IFN-γ PE, anti-CD45RA BV605, anti-CD62L BV421, anti-CD56 AF700, anti-CD33 APC, anti-CD19 PE-Cy7 mAbs, and 7-AAD (all BD Biosciences). After staining and lysis of erythrocytes, samples were acquired on a BD FACSCanto 10c with at least 10,000–50,000 events in the CD45⁺ leukocyte gate.