Cxcr4 12g5

Neutrophils were suspended in ice-cold phosphate-buffered saline with 1% fetal bovine serum at 2 × 10⁶ cells/ml. Antibodies used in the experiment include CD66b (G10F5, BD Pharmingen, USA), CD10 (HI10α, Biolegend, USA) CXCR4 (12G5, Biolegend, USA) and P2X1 (APR-022-F, Alomone labs, Israel). Apoptosis was quantified by annexin V-PE and propidium iodide (PI) staining, with early apoptotic cells being annexin V⁺/PI^- and late apoptotic cells annexin V⁺/PI⁺. We counted the proportion of all apoptotic neutrophils (annexin V⁺). Antibodies or apoptosis kits were used according to the manufacturer’s instructions. After incubating for 20 min at 4 °C in the dark, cells were washed and measured using a FACS Canto II cytometer (BD Biosciences). The data were analyzed using FlowJo V10 (Ashland, OR, USA).

NK cells were stained with antibodies recognizing CD56 (NCAM16.2), CD3 (UCHT1), CD34 (581), CD4 (RPA-T4), CD19 (HIB19), TNF-α (Mab 11), and IFN-γ (B27) from BD Biosciences (Franklin Lakes, NJ, USA), CD107a (H4A3), CD34 (561), and CXCR4 (12G5) from BioLegend (San Diego, CA, USA), along with propidium iodide (BD Biosciences), annexin V (BioLegend), and/or a Live/Dead fixable aqua dead cell stain kit (Thermo Fisher Scientific). Cells were examined on a BD Biosciences LSRFortessa instrument, and data were analyzed using FlowJo v10.1 (BD Biosciences). Statistical analyses were performed with GraphPad Prism 8.4.1.