Tlr9 fitc

The expression of surface markers on MSC, nvMSC-HLA-G1, and vMSC-HLA-G1 was evaluated by flow cytometry using the following monoclonal anti-human antibodies: HLA-G FITC (ABD Serotec, Oxford, UK), HLA DR, DP, DQ FITC (BD Bioscience, San Jose, CA), HLA-ABC PE (BD Pharmingen, San Jose, CA), MICA/MICB FITC, CD155 FITC (both from ABD Serotec), ULBP-1 PE, ULBP-2 PE (both from R&D Systems), CD58 PE (BD Biosciences), TLR9 FITC, TLR4 PE (both from Abcam, Cambridge, UK) and TLR3 Alexa 647 (Imagenex, Port Coquitlam, BC, Canada). Cells were also stained for HLA E (Abcam), ULBP-3 (R&D Systems), and CD112 (ABD Serotec) using Alexa Fluor 488 Goat Anti-Mouse IgG (Molecular Probes-Life Technologies) as secondary antibody. Briefly, cells were incubated for 15 minutes in the dark with saturating concentrations of each antibody. Stained cells were then washed with phosphate-buffered saline 0.1% azide, fixed with 1% formaldehyde, and analyzed by flow cytometry using the CellQuest software (Becton Dickinson Biosciences, San Jose, CA). A total of 10,000 events was acquired for each sample. Appropriate isotype (negative) controls were performed for each antibody.