Fitc conjugated anti human cd14 antibody

We isolated CD4⁺ T helper cells from PBMCs according to the manufacturer’s instructions (https://www.miltenyibiotec.com/_Resources/Persistent/3c804fa07b66b63215bbacbf43387804b151d77f/SP_CD4.pdf). Venous blood samples were collected from the patients with ASO or the healthy donors at the First Affiliated Hospital of Sun Yat-sen University, and PBMCs were isolated through Ficoll centrifugation (GE Healthcare, Catalog #17144002). CD14⁺ cells were isolated from PBMCs with CD14 magnetic bead kits (Miltenyi, Catalog# 130-050-201) and FITC-conjugated anti-human CD14 antibody (BD, Catalog# 555397) according to the manufacturer’s instructions. CD4⁺ T cells were isolated from the rest of the CD14⁻ cell suspensions by positive selection with a CD4 magnetic bead kit (Miltenyi, Catalog# 130-045-101) and PE-conjugated anti-human CD4 antibody (BD, Catalog# 555347) according to the manufacturer’s instructions. Isolated CD4⁺ T cells, CD14⁺ cells and CD4⁻CD14⁻ cells were validated by FACS and frozen in liquid nitrogen for future experiments.

PBMC were stained with FITC-conjugated anti-human CD14 antibody (BD Pharmingen), and PE-conjugated anti-CD1d antibody (BD Pharmingen) or isotype control (BD Pharmingen). Cells were then fixed and acquired by flow cytometry. Monocytes were gated based on their characteristic appearance on FSC/SSC dotplot and upon CD14 expression. The level of CD1d expression was expressed as the difference in mean fluorescence intensity (DMFI) between CD1d staining and isotype control of each sample as baseline.

Membrane expressions of CD86, CD163, CD80, and CD206 were measured in human monocytes and Raw 264.7 cells after being washed in buffer containing 0.1% bovine serum albumin (BSA). Monocytes were gated on the basis of forward and side light scatter and by using a fluorescein isothiocyanate (FITC)-conjugated anti-human CD14 antibody (BD Bioscience) and Fixable Viability Dye (Thermo Fisher Scientific). The following antibodies were used for staining: allophycocyanin (APC)-conjugated anti-human CD86 (BD Bioscience), peridinin chlorophyll protein/cyanin 5.5 (PerCP/Cy5.5)-conjugated anti-human CD163 (BD Bioscience), phycoerythrin (PE)-conjugated anti-mouse CD80 (BD Bioscience), and APC-conjugated anti-mouse CD206 (Thermo Fisher Scientific). Cells were stained in the dark at 4 °C for 30 min. All flow cytometry data were acquired on a BD FACScanto flow cytometer (BD Bioscience) and analyzed by FlowJo software (TreeStar, Inc., Ashland, OR, USA).