Biotin conjugated mouse anti human cd14 monoclonal antibody

To examine the role of TNF-α in regulating Mincle expression, synovial fibroblasts and macrophages were extracted from the ST from the knees of 10 KOA patients. Following collagenase digestion of ST, the extracted cells were incubated with biotin-conjugated mouse anti-human CD14 monoclonal antibody (1 : 20; clone M5E2, Biolegend) for 30 minutes at 4°C. The cells were subsequently washed twice with PBS, combined with streptavidin-conjugated magnetic particles (BD Biosciences, CA, USA), and separated in a magnetic separation system (BD IMag^TM cell separation system, BD Biosciences) into CD14⁺ and CD14^- cells, as described elsewhere [13 (link)]. CD14⁺ and CD14^- cell fractions from five patients were immediately analyzed for TNF-α, CD14, and Mincle expression using quantitative polymerase chain reaction (qPCR), while those from the remaining five patients were cultured for 7 days in six-well plates containing α-MEM. The cells were subsequently stimulated with vehicle (α-MEM), 10 ng/ml human recombinant TNF-α (Biolegend), or 10 ng/ml TNF-α + 1 µg/ml anti-TNF-α antibody (clone Mab11, Biolegend) for 24 hours, before isolating RNA for real time (RT)-PCR analysis for Mincle expression.

To examine MINCLE expression in macrophages, we extracted synovial macrophages from the ST of the knees of 5 OA and 5 RA patients. ST was digested with collagenase and the resulting cells were incubated with biotin-conjugated mouse anti-human CD14 monoclonal antibody (1 : 20; clone M5E2, BioLegend) for 30 minutes at 4°C. After washing twice with phosphate-buffered saline (PBS), the cells were added to streptavidin-conjugated magnetic particles (BD Biosciences, CA, USA) and separated in a magnetic separation system (BD IMag cell separation system, BD Biosciences) into CD14+ (macrophage) and CD14– cells, as described elsewhere [13 (link)]. Freshly extracted CD14+ and CD14– cell fractions were subjected to qPCR to analyze CD14 and MINCLE expression in OA and RA.