Anti cd14 fitc

Complete media (CM) including RPMI 1640 (Gibco, USA, NY) that contains 10% Fetal Bovine Serum (FBS) (Gibco, USA, NY), Streptomycin 100 μg/mL, Penicillin 100 IU/mL (Gibco, USA, NY), 2 mmol/L of L-glutamine (Gibco, USA, NY). 2-mercaptoethanol (2ME) was ordered from Gibco (USA, NY). Recombinant human granulocyte macrophage colony stimulating factor (rh GM-CSF) was purchased from Sigma Chemical Co (Munich, Germany) and recombinant human interleukin-4 (rh IL-4) from eBioscience (CA, USA). Lipopolysaccharide (LPS) was ordered from Sigma Chemical Co (Munich, Germany). Carboxyfluorescein succinimidyl ester (CFSE) cell labeling kit was obtained from BioLegend (San Diego, United States). Human pan T cell isolation Kit was purchased from MiltenyiBiotec, Germany. Antibodies used to phenotype the cells were anti-HLA-DR-APC and anti-CD86- PerCP-cy5.5 from BioLegend (San Diego, United States), anti-CD40-CF-blue, anti-CD11c-FITC, and anti-CD14-FITC from Immunostep (Salamanca, Spain). Ficoll was obtained from Sigma Chemical Co (Munich, Germany). Bradford protein assay kit was purchased from Bio-Rad, (Hercules, CA).

For immunophenotypic characterization of cells, 1.0 × 10⁶ BMSCs per tube were washed with FACS buffer consisting of PBS supplemented with 1% bovine serum albumin (BSA, Sigma). Then, the cells were incubated for 30 minutes in the dark at room temperature with the following fluorochrome-conjugated primary antibodies: anti-CD90-Percp-Cy5.5, anti-CD73-APC, anti-CD105-FITC, anti-CD146-PE (all from BioLegend, San Diego, CA, USA), anti-CD14-FITC (Immunostep, Salamanca, SPA), anti-CD34-FITC, anti-CD45-Percp-Cy5.5 (both from Agilent DAKO, Santa Clara, CA, USA), and anti-CD11b-PE (Santa Cruz Biotechnology, Dallas, TX, USA). The isotype controls were IgG2A-FITC, IgG1A-APC, IgG1A-Percp-Cy5.5, IgG1-PE, IgG1-FITC, and IgG2A-PE (all from Santa Cruz Biotechnology). Next, the cells were washed with FACS buffer and resuspended in 300 μL buffer for acquisition with a BD FACSCanto™ cytometer (BD Biosciences). The data were analyzed with FlowJo software (Tree Star, Ashland, OR, USA).