Percp vio700 mouse igg1 isotype

After different neutrophil subgroups were obtained from step 2.2.1.2, typically 1 × 10⁵ cell/50 μl and the following anti-human fluorochrome-conjugated mAbs or specific isotype controls: CD66b (Biolegend, USA), PE-Cy7 mouse anti-human CD10 (Biolegend, USA), PerCP-vio700 mouse anti-human CD11b (Miltenyi, German), APC mouse anti-human CD16 (Biolegend, USA), PE mouse IgG₁κ isotype control (Biolegend, USA), PE-Cy7 mouse IgG₁κ isotype control (Biolegend, USA), PerCP-vio700 mouse IgG₁ Isotype (Miltenyi, German), and APC mouse IgG₁κ isotype control (Biolegend, USA) 10 μl, respectively were intubated for 15 min in the dark room at room temperature, 500 μl RBC Lysis Buffer (Macs, German) was added into the tube. The mixture was centrifuged with 2,000 rpm for 5 min after another intubating for 15 min in the dark room at room temperature, then removed the supernatant and added 500 μl PBS fluid into the precipitate to resuspend cells which was analyzed by eight-color three-laser-MACSQuant Analyzer (Miltenyi Biotec, German) while data analysis performed using FlowJo software (Tree Star, USA).

Added 100 μl peripheral anticoagulant blood in healthy volunteer or sepsis patient into the tube with following anti-human fluorochrome-conjugated mAbs or specific isotype controls: PE mouse anti-human CD66b (Biolegend, USA), PE-Cy7 mouse anti-human CD10 (Biolegend, USA), PE mouse IgG₁κ isotype control (Biolegend, USA), PE-Cy7 mouse IgG₁κ isotype control (Biolegend, USA), PerCP-vio700 mouse IgG₁ Isotype (Miltenyi, German), and APC mouse IgG₁κ isotype control (Biolegend, USA) 10 μl, respectively. After intubating for 15 min in the dark room at room temperature, 500 μl RBC Lysis Buffer (Macs, German) was added into the tube. The mixture was centrifuged with 2,000 rpm for 5 min after another intubating for 15 min in the dark room at room temperature, then removed the supernatant and added 500 μl PBS fluid into the precipitate to resuspend cells which was analyzed by eight-color three-laser-MACSQuant Analyzer (Miltenyi Biotec, German) while data analysis was performed by using FlowJo software (Tree Star, USA).