Pe cy7 conjugated anti human cd15

Supernatants of CXCR4^lo and CXCR4^hi neutrophils were collected after 6-hour cell culture, and lactate was assessed via a lactate assay kit (A019-2-1, Nanjing Jiancheng Bioengineering Institute, Nanjing, China) according to the manufacturer’s instructions. For glucose uptake detection, isolated neutrophils were incubated with 100 mM 2-NBDG (N13195, Invitrogen) for 2 h and then stained with PE-Cy7 conjugated anti-human CD15 (301924, 1:100, BioLegend) and PE conjugated anti-human CXCR4 (306506, 1:100, BioLegend) at 4 °C for 30 min before measuring fluorescence by flow cytometry.

To assess the phagocytic capacity, neutrophils from healthy controls and psoriasis patients were isolated and incubated with pHrodo Green E coli (2 mg/mL; P35366, Thermo Fisher Scientific, USA) at 37 °C for 30 min. Incubation was stopped by the addition of 2 mL of PBS, and cells were then washed 3 times. Cells were divided into two parts, the first of which was used for flow staining. After incubated with PE-Cy7 conjugated anti-human CD15 (301924, 1:100, BioLegend) and PE conjugated anti-human CXCR4 (306506, 1:100, BioLegend) at 4 °C for 30 min. Phagocytic uptake was analyzed by flow cytometry and expressed as median fluorescence intensity (MFI). The remaining cells, which were not used for flow staining, were incubated with Hoechst 33258 (C0021, 1:1000, Solarbio technology) for 15 min for following confocal microscope (LSM880, Carl Zeiss, Germany).