Pe anti human cd45 antibody

Cell pellets were collected by spinning at 200 × g for 5 min at 4 °C. Cells were re-suspended in 250 µl blocking agent (PBS/0.2% BSA/0.1% sodium azide) and stained with either IgG control (PE Mouse IgG1 Isotype Ctrl -Biolegend-400111) or the antibody stained for its corresponding antigen CD45 (PE anti-human CD45 Antibody Biolegend-304008) at 4 °C in the dark for 60 min. Five milliliter buffer was then added, and samples were spun at 200 × g for 5 min at 4 °C. Buffer was discarded, and the cells were re-suspended in 300 µl PBS and kept at 4 °C in dark until analysis. Mean fluorescent intensity was quantified using the LSR II flow cytometer (BD Biosciences-UK) and data acquisition was performed with FACSDiva software.

THP-1 cells namely the human monocytic leukemia cell line (acquired from China National Collection of Authenticated Cell cultures) were differentiated with phorbol 12-myristate 13-acetate (PMA #HY-18739, MedChemExpress, Shanghai, China) with a concentration of 200 ng/mL for two days in RPMI 1640 medium containing 10% FBS. When THP-1 reached confluence, the medium was supplemented with SAAs (#ab50232, Abcam) with a concentration of 500 ng/mL or SAAs of 500 ng/mL combinding with TLR2 inhibitor TLR2-IN-C29 (#S6597, Selleck, Shanghai, China) of 20 µM or CXCR2 inhibitor for 12 h. Collected THP-1 cells were stored in cell staining buffer (#420201, Biolegend, San Diego, CA, USA) and incubated with Zombie NIR™ Fixable Viability Kit (#423106, Biolegend), PE anti-human CD45 antibody (#368510, Biolegend) and PE/Cyanine7 anti-human CD86 antibody (#374210, Biolegend). After fixation and permeabilization of cells using BD Cytofix/Cytoperm™ Fixation/Permeabilization Solution Kit (#B554714, BD Biosciences Pharmingen, San Diego, CA, USA), BV421 anti-human CD68 Y1/82 A (#564943, BD Biosciences Pharmingen) and APC anti-human CD206 (MMR) antibody (#321110, Biolegend) were then applied to stain intracellular markers.