Anti cd45 pecy7

Anti-CD45-PeCy7, anti-CD11b-PeCy5, anti-CD166-phycoerythrin (PE), anti-CD105-PE, anti-CD90-PeCy5, anti-CD34-PE, isotype control fluorescein isothiocyanate (FITC) human IgG1, and isotype-control PE human IgG2a were purchased from Beckman Coulter (Indianapolis, IN). Anti-CD44-allophycocyanin (APC) was purchased from BD Biosciences (San Jose, CA). Type 1 collagenase, bovine serum albumin (BSA, fraction V), calcium chloride, cetylpyridinum chloride (CPC) dexamethasone, isobuytlmethylxanthine, indomethacin, ascorbate 2-phosphate, β-glycerol phosphate, alizarin red s, and oil red o were purchased from Sigma (St. Louis, MO).

Three milliliters of PBMCs with a concentration of 5 × 10⁶ cells/ml was cultured in a well of a 6-well plate. After 1 h of incubation at 37°C and 5% CO₂, the nonadherent cells were washed 3 times with warm PBS. The remaining adherent monocytes were prestimulated for 24 h with concentrations of different ligands that would induce either training or tolerance, such as β-glucan (1 μg/ml), MDP (1 μg/ml), flagellin (1 μg/ml), R848 (1 μg/ml), Pam3CSK4 (1 μg/ml), and RPMI (negative control). The stimuli were washed away and the cells were further incubated for 5 days in the presence of RPMI supplemented with 10% pooled human serum. On day 7, the cells were incubated for 1 h with Versene solution, collected, harvested by centrifugation, and suspended in PBS supplemented with 1% protein blocking agent (PBA). The cells were washed two times and stained extracellularly with the following antibodies: anti-CD45-PeCy7 (Beckman Coulter), anti-CD68-APC (BioLegend), anti-CD14-PE (Beckman Coulter), anti-CD11b-FITC (Beckman Coulter), and anti-DC-SIGN-APC (BioLegend). The samples were measured on a fluorescence-activated cell sorter (FACS) FC500, and the data were analyzed using the CXP software (Beckman Coulter).

Anti-CD45-PeCy7, anti-CD11b-PeCy5, anti-CD166-phycoerythrin, anti-CD105-phycoerythrin, anti-CD90-PeCy5, anti-CD34-phycoerythrin, isotype control fluorescein isothiocyanate human IgG₁, and isotype-control phycoerythrin human IgG_2a were purchased from Beckman Coulter (Indianapolis, IN, USA). Anti-CD44-allophycocyanin was purchased from BD Biosciences (San Jose, CA, USA). Type 1 collagenase, bovine serum albumin (fraction V), calcium chloride, dexamethasone, isobuytlmethylxanthine, indomethacin, ascorbate 2-phosphate, β-glycerol phosphate, alizarin red S, oil red O, cetylpyridinium chloride, BCIP/NBT, silver nitrate, E2, daidzein, and genistein were purchased from Sigma (St Louis, MO, USA). daidzein analogs (i.e. 2c, 2g, and 2l) were synthesized in our laboratory as described previously [21 (link), 22 (link)].