Lsr fortessa flow cytometry machine

Peripheral blood was collected in EDTA vial, and 200 μl of blood was aliquoted for antibody staining. Briefly, 1 ml of RBC lysis buffer was added to 200 μl of whole blood, and mixed properly. A 10× red blood cell lysis buffer was prepared in-house using NH₄Cl (0.155 M), KHCO₃ (0.01 M) and EDTA (0.1 mM). The tubes were incubated for 10 min at 4 °C, and centrifuged at 300×g for 10 min. The cell pellets were washed twice with 1× PBS and suspended in 300 μl of PBS to obtain a single cell suspension. For cell surface staining, 100 μl of cell suspension was used, and the antibodies CD16 (1:100) (clone 3G8, APC conjugated, Cat No 302011, Biolegend, USA), CD66b (1:100) (clone G10F5, FITC conjugated, Cat No 305103, Biolegend, USA) and CD177 (1.5:100) (clone MEM-166, APC/Cyanine 7 conjugated, Cat No 315809, Biolegend, USA) was used. Unstained controls, stained samples, and fluorescence minus one controls were acquired on BD-LSR Fortessa flow cytometry machine. Using the FACS DIVA software, PMN gating was done based on FSC-A v/s SSC-A plot. Enriched and activated neutrophils were gated based on CD16⁺ CD66b⁺ (double positive) in a quadrant plot. CD177⁺ neutrophils were gated within these double positive cells.

Human PBMCs were separated from whole blood (from the internal donor program at Novartis) using Leucosep tubes, and monocytes were isolated following the Miltenyi Biotech Classical Monocyte Isolation Kit. Cells were plated in a 96-well round bottom plate at a concentration of 5 × 10⁴ cells/well and were incubated for 7 days with 50 ng/ml M-CSF at 37°C. On day 7, various concentrations of sabatolimab (or hIgG4) were added to the cells for 10 minutes at RT, followed by addition of 5 × 10⁴ cells (100 μl) of CFSE-labeled target cells (SKM-1 or HNT-34). Plates were incubated for 4 hours at 37°C at 5% CO₂. After 4 hours, cells were washed with PBS, stained for CD11c, and run on a BD LSR Fortessa flow cytometry machine. Data were analyzed using FlowJo analysis software and graphed using GraphPad Prism. ADCP activity was determined as percentage of CFSE⁺CD11c⁺ events.