Blood sample collection and sample measurement
hemolysis, TNF-α
LYSED, STERILE, HEMOLYSIS
Before and 24 h after the procedure, 2 mL of venous blood was drawn, placed in sterile EDTA tubes for flow cytometry analysis and cytokine testing. 100 uL of blood was added with 5 uL each of fluorescein antibodies PE anti-human CD11b (Cat No. 301306, Lot No. B338581, Biolegend, USA), PE/Cyanine7 anti-human CD66b (Cat No. 305116, Lot No. B345422, Biolegend, USA) and FITC anti-human CD64 (Cat No. 399506, Lot No. B357054, Biolegend, USA), and another 100uL of blood was added with antibodies as a control group. Specimens were incubated for 30 min at room temperature, protected from light, and erythrocytes were lysed using EPICS hemolysis instrument (Beckman Coulter Company, USA). Flow cytometry (Beckman Coulter FC 500MPL, USA) was used for detection and analysis.The remaining venous blood was centrifuged at 3000 g for 30 min at 4 °C, and then the plasma was collected. Plasma was equated into polypropylene tubes and stored in a refrigerator at -80 °C for cytokine detection. Plasma concentrations of the inflammatory cytokines IL-1β (Cat No. CHE0001, Lot No. 20220920, Beijing 4A Biotech Co., Ltd, China), IL-6 (Cat No. CHE0009, Lot No. 20221008, Beijing 4A Biotech Co., Ltd, China), TNF-α (Cat No. CHE0019, Lot No. 20220920, Beijing 4A Biotech Co., Ltd, China) and S100β (Cat No. E-EL-H1297c, Lot No. AK03886L3479, elabscience, China) were measured using commercial enzyme-linked immunosorbent assay (ELISA) kits following the manufacturer’s instructions. ELISA test was read by URIT-660 Microplate Reader and the testers were unaware of the clinical information.