Secondary donkey anti rabbit fitc

Adherent cells were harvested using Cell Dissociation Buffer (Invitrogen) or TrypLE (iPS cells) fixed in 4% paraformaldehyde (Tousimis), rinsed 2Χ with PBS, blocked using 0.5% donkey serum (Fitzgerald) and 1% bovine-serum albumin (Sigma) for 1 hour at room temperature (RT), and permeabilized (if needed) with 0.7% Tritron X-100 (MP Biomedicals) in PBS. For Stage 1, cells were stained with either 1:100 Alexa Fluor 647^®-conjugated anti-Flk-1 antibody (Biolegend) or 1:50 Flk-1 rabbit polyclonal antibody (Santa Cruz Biotechnology) followed by 1:100 secondary donkey anti-rabbit FITC (Biolegend) or 1:75 anti-human CD309/VEGFR2 PE monoclonal (Biolegend) and BD Biosciences 1:1000 human Fc Block (1:1000) followed by 1:75 mouse IgG1, κ°PE (Biolegend) was used for the isotype control. For Flt-1 expression on mouse VPC, cells were stained with goat IgG anti-Flt-1 (Santa Cruz) or goat IgG (Abcam) for one hour followed by anti-goat FITC (Santa Cruz) at 1:200. Stage 2, cells were stained with CD144 (VE-cadherin) PerCP-efluor710^® at 1:200. All cells were counterstained with 1:1000 eFluor 780 Fixable Viability Dye (eBioscience) and analyzed on an LSR II Flow Cytometer System (BD Biosciences).

MoDCs were cultured on 8-well chambered glass slides (ThermoScientific, Pittsburgh, PA). After 20 min treatment with PA or PA+CLI-095, the MoDCs were fixed and stained with primary anti-human phospho NF-κBp65 monoclonal antibodies overnight at 4°C (Cell signaling Technologies, Danvers, MA) and secondary donkey anti-rabbit FITC (Biolegend). Slides were sealed after addition of mounting media with DAPI and photos were taken at 40x magnification on a Keyence BZ-9000 fluorescent microscope.