Synovial samples collected from each hip joint during THR were digested with type I collagenase for 24 h at 37°C to isolate mononuclear cells. After reacting with 0.5 ml biotin-conjugated anti-CD14 human monoclonal antibody (BioLegend) for 30 min at 4°C, the cells were washed twice in phosphate-buffered saline (PBS) and reacted with streptavidin-conjugated magnetic beads (BD Biosciences). The cells were subsequently transferred to a cell separation system (IMag; BD Biosciences) and left to stand for 30 min at 4°C. After removal from the magnetic support, 5 ml of prewarmed (37°C) α-minimum essential medium (MEM) was added, and the cells were centrifuged at 280 g for 5 min to isolate CD14+ (macrophage-rich fraction) and CD14- (fibroblast-rich fraction) cells. RNA sample concentrations were measured twice using the DeNovix DS-11 Spectrophotometer (DeNovix Inc., Wilmington, DE, USA) and adjusted to a concentration of 100 ng/μl; a total of 1 μg of RNA was used for cDNA synthesis. NGF, CD14, and CD90 mRNA expression in CD14+ and CD14- cells isolated from 8 patients were separately examined using qPCR. NGF expression (NGF/GAPDH) in CD14+ and CD14- cells was determined using the delta-delta CT method. When the average NGF expression (NGF/GAPDH) level in CD14- cells was 1, the relative expression (CD14+ cells/CD14- cells) was calculated.
Biotin conjugated anti cd14 human monoclonal antibody
Manufactured by BioLegend
Biotin-conjugated anti-CD14 human monoclonal antibody. Core function is to detect CD14 expression on human cells.
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Lab products found in correlation
2 protocols using biotin conjugated anti cd14 human monoclonal antibody
1
Isolation and Quantification of NGF Expression in Synovial Macrophages and Fibroblasts
2
Isolation and Culture of CD14+ Monocytes from Synovial Tissues
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Patients’ synovial tissues were treated with 20 ml of 0.1% type I collagenase from Clostridium histoluticum (Wako Pure Chemicals, Osaka, Japan) for one day at 37°C. Isolated mononuclear cells were then incubated with 0.5 ml of biotin-conjugated anti-CD14 human monoclonal antibody (BioLegend) for 30 min at 4°C, before washing twice in phosphate-buffered saline (PBS). The cells were subsequently exposed to streptavidin-conjugated magnetic beads (BD Biosciences) and moved to a cell separation system (IMag; BD Biosciences) where they were left to stand at 4°C. Following a period of 30 min, the cells were removed from the magnetic support. To extract CD14+ cells, 5 ml of prewarmed (37°C) α-minimum essential medium (MEM) was added before centrifuging the cells at 280 g for 5 min. After trypan blue staining, CD14+ cells (1 × 105 cells/5 ml) were seeded in a 6-well culture plate.
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