Phycoerythrin pe conjugated streptavidin

For this analysis, peripheral blood was collected from the tail vein of hNOJ mice. Human Flt3-L and GM-CSF in the plasma of hNOJ mice was determined by cytometric bead array (CBA) (33 (link)). Plasma samples were serially diluted with the Assay Diluent supplied in the BD CBA Human Soluble Protein Master Buffer Kit (BD Biosciences, San Diego, CA, USA). The Human GM-CSF Flex Set (BD Biosciences) was used to measure circulating human GM-CSF. To measure circulating human Flt3-L, an anti-human Flt3-L monoclonal capture antibody (40416; R&D Systems, Minneapolis, MN, USA) was conjugated to beads (Functional Bead A9; BD Biosciences) that have a distinct fluorescent intensity from the human GM-CSF capture bead using the Functional Bead Conjugation Buffer Set (BD Biosciences). A biotinylated anti-human Flt3-L polyclonal antibody (R&D Systems) was used as a detection antibody and was treated with phycoerythrin (PE)-conjugated streptavidin (BioLegend, San Diego, CA, USA). Data were collected on a FACSCanto II (BD Biosciences) and analyzed using FCAP Array Software v3.0 (Soft Flow Inc., St. Louis Park, MN, USA) based on the fluorescent intensity of PE. Standard curves were set using recombinant human Flt3-L (R&D Systems) and GM-CSF (supplied in the Human GM-CSF Flex Set). The limit of detection of both human Flt3-L and GM-CSF was <0.01 ng/ml.