Anti human cd14 efluor450

Lithium-heparinized blood from BALB/c WT and Ncf1** mice or human NHD and SLE/CGD patients was mixed with SNECs in a 1:2 ratio and incubated at 37 °C. In some experiments, the blood was preincubated with varying concentrations of H₂O₂ (10 min), of the aminoferrocene compound MIS43 (10 min) [32 (link)], varying concentrations of catalase (Sigma) or butylated hydroxyanisole (BHA, Sigma) for 15 min before adding SNECs. Cells were stained with anti-mouse CD11b-eFluor450 (eBioscience), anti-mouse Ly6C-APC (BioLegend), Ly6G-PE/Cy7 (BioLegend), CCR2-FITC (BioLegend), I-A/I-E (MHC II)-Alexa Fluor 700 (BioLegend) or anti-human CD14-eFluor450 (eBioscience), CD16-APC/Cy7 and CCR2-PerCP/Cy5.5 (BioLegend), respectively. Samples were subjected to hypotonic water lysis of erythrocytes and analyzed using a Beckman Coulter Gallios™ flow cytometer. The Phagocytosis index (PhIx) was calculated in the following way: PhIx = (%PI/pHrodo-positive phagocytes x MFI^PI/pHrodo). For the analysis of cytokine/chemokine production upon phagocytosis, plasma was isolated from blood incubated with SNECs (3 h) and subjected to LegendPlex bead-based assay (BioLegend).

For measurement of intracellular ROS in human phagocytes, lithium-heparinized blood was incubated with dihydrorhodamine (DHR) 123 (3 μg/ml, Molecular Probes) for 15 min at 37 °C. Cells were stained with anti–human CD14-eFluor450 (eBioscience, cat.# 48–0149, clone 61D3) and CD16-APC/Cy7 (Biolegend, cat.# 302017, clone 3G8), and anti-human CD192/CCR2 (Biolegend, cat.# 357203, clone K036C2), and ROS production was induced by incubation with PMA (100 ng/ml) for 15 min at 37 °C. Intracellular ROS in mouse CD11b⁺Ly6C^HI iMonos was measured via DHR123 fluorescence after 3 h incubation of mouse blood with various concentrations of MIS43 or RE-02 or 15 min incubation with 100 ng/ml PMA. Before analysis on the Beckman Coulter Gallios™ flow cytometer, human and murine samples were subjected to hypotonic water lysis.