Helix np nir

Target YAC‐1 cells were labelled with carboxyfluorescein succinimidyl ester (CFSE; C1031, Beyotime) at a final concentration of 5 μM. They were then co‐cultured with effector NK cells at an effector‐to‐target cell (E:T) ratio of 2:1 or 0:1 for 4 h at 37°C under 5% CO₂. After 4 h, the samples were stained with Helix NP NIR (425301, BioLegend) and Annexin V‐PE (640908, BioLegend) for 10 min. Fluorescence‐activated cell sorting (FACS) analysis was performed immediately with a BD LSR Fortessa Cell Analyzer (BD Biosciences). The Incucyte S3 Live‐Cell Analysis Instrument (Sartorius AG) was used to monitor the killing activity of NK cells. Propidium iodide (C1052, Beyotime) was used to mark dead cells.

ROS production was measured by flow cytometry using dihydrorhodamine 123 (DHR123). Cells were preincubated for 20 minutes at 37 °C. The indicated agonists and DHR (2 µM) were then added for 15 minutes. To stop the reaction, cells were placed on ice, washed, and resuspended in Cell Wash (BD Bioscience) containing 0.5% PFA and 50 nM of the cell-impermeant far-red emitting nucleic acid stain Helix NP NIR (Biolegend) to gate for live cells. 10,000 live cells per condition were analysed on a Guava easyCyte™ System (Millipore, Burlington, MA, USA) for median fluorescence intensity. Agonist-mediated ROS production was expressed as percentage of the signal detected in untreated cells. The FPR2/ALX specific agonist WKYMVm was used as a positive control.

Plated human iPSC-derived NPCs (hNPCs) were harvested using Accutase cell dissociation solution (GIBCO), washed in ice-cold flow buffer (DPBS, 2% fetal bovine serum, 2 mM EDTA). In each condition, 1 × 10⁶ cells were incubated with the NSC-6 antibody for 45 min on ice (1:20; optimal concentration determined by previous titration experiments). The cells were then washed twice in ice-cold flow buffer with 2% normal donkey serum added, then stained in the same buffer with Alexa Fluor 405 (1:200 donkey anti-mouse IgG H&L; ABCAM 175659) secondary antibody for 45 min on ice. Stained cells were washed twice in ice-cold flow buffer then resuspended in a viability dye solution (20 mM Helix NP NIR, BIOLEGEND, 425301 in flow buffer) and immediately analyzed by flow cytometry using an LSR Fortessa (BECTON DICKINSON) with acquisition of AF405 on V450 and Helix-NIR on R670. Final data analyses were performed using FlowJo software (TREE STAR INC.). To determine hNPC NSC-6 positivity, 250,000 live cell events were acquired. NSC-6 positive signal was set by gating for single viable cells and above background, the latter determined using the signals obtained from unstained hNPCs and hNPCs stained with the secondary antibody only.