Goat anti mouse fitc

BoMac cells were transiently transfected with cloned full length wild type CD1b1, CD1b3, and CD1b5 in pcDNA3.1 using Fugene-6 reagent (Roche) according to the manufacturer’s protocol, and analysed 48 hours after transfection. K562 cells transfected with bovine CD1d and human CD1a, and C1R cells transfected with bovine CD1b and CD1a were described previously [3 (link), 12 (link)]. The anti bovine CD1 antibodies CC20 (IgG2a), CC43 (IgG2b), and CC122 (IgG1) were kindly provided by Dr. C. J. Howard, Compton, UK; 20.27 SBU-T6 (IgG1) was obtained from the European Collection of Cell Cultures; CC14 (IgG1) and CC118 (IgG1) were kindly provided by Dr. J. C. Hope, Compton, UK; BCD1b.3 (IgG1) was provided by Dr. D. B. Moody, Brigham and Women’s Hospital, Boston, goat anti mouse FITC and goat anti mouse PE were obtained from Becton Dickinson. PE-labelled anti bovine Ig light chain antibody IL-A59 was used as B cell marker (Serotec).

Cells were harvested gently from non-adherent dishes, washed with PBS (w/o Ca²⁺; Mg²⁺) and resuspended in PBS (w/o Ca²⁺; Mg²⁺)/0.5% BSA to yield 5x10⁶ cells/mL. Flow-cytometric analysis of surface exposed ceramide of 5x10⁵ cells was performed. Cells were incubated for 15 minutes on ice with saturating concentrations of the antibodies against ceramide (MID15B4, Alexis), CDw17 (G035-FITC, Becton-Dickinson), CD65s (VIM2-FITC, Caltag) and CD77 (38–13, Immunotech). Monoclonal antibodies used for detection were directly fluorochrome-conjugated or indirectly labeled by subsequent incubation with fluorochrome-conjugated secondary reagents (ceramide: goat anti mouse-FITC, Becton-Dickinson; CD77: goat anti rat-PE, Dianova) after two washing steps with PBS containing 0.5% BSA. GM1 was stained using Alexa Fluor 488 labeled cholera toxin subunit B (Molecular Probes).

Purified exosomes (200 μg) were incubated with 10 μL of 4-μm-diameter aldehyde/sulfate latex beads (Invitrogen, Eugene, OR) in PBS for 1 h at room temperature with gentle agitation. After washing with PBS, samples were blocked with 200 mM glycine for 30 min and washed again. Beads were incubated with the following exosome-associated antibodies for 30 min: anti–CD63 (Millipore, Billerica, MA, USA), anti-CD9 (BD Biosciences, San Jose, CA, USA), anti-HLA-A,B,C (BioLegend, San Diego, CA, USA), anti-TSG101 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-HSP70 (Novus Biologicals, Littleton, CO, USA), anti-lysosomal membrane glycoproteins CD107a (LAMP-1) (Miltenyi Biotec, Auburn, CA, USA), anti-MICA/B (eBioscience, Grand Island, NY, USA), anti-CD81 (ProSci, Fort Collins, CO, USA), anti-TGF-β (Peprotech, Rocky Hill, NJ, USA), and anti-FasL (Boster, Pleasanton, CA, USA). Staining with secondary antibodies for 30 min followed, using goat anti-rabbit Alexa Fluor 488 (Invitrogen, Eugene, OR, USA) and goat anti-mouse FITC (Becton Dickinson, Franklin Lakes, NJ, USA). A “bead-only” control as well as isotype-matched antibody controls were also prepared. Samples were washed twice, fixed with 1% paraformaldehyde and analyzed using a MACSQuant Analyzer and FlowJo software. Single beads were gated for fluorescence analysis.