Apc h7 conjugated anti cd45

Cell suspensions were stained with fluorophore-conjugated monoclonal antibodies. The following antibodies were used for cell surface staining of mononuclear cells derived from blood and tumors of patients: PE-Cy7-conjugated anti-CD24, APC-conjugated anti-CD44, APC-H7-conjugated anti-CD45, PE-conjugated anti-CD49f, FITC-conjugated anti-CD227, PerCP-Cy5-conjugated anti-CD326 (EpCAM), and PE-conjugated anti-CD309 (all Becton Dickinson, San Jose, CA, USA). The following isotype control groups were used: PerCPCy5.5 IgG1, APC IgG2b, APC-H7 IgG2b, PE IgG2a, FITC IgG1, and PE-Cy7 IgG1. All antibodies were titrated to determine their optimal staining concentration and appropriate isotype controls were used. Labeled cells were washed thoroughly with 500 μL of FACSFlow (Becton Dickenson, Franklin Lakes, NJ, USA, Cat# 342003).
All samples were run on a Becton Dickenson FACSCanto II flow cytometer. The instrument was set up and standardized using BD Cytometer Setup and Tracking (CS&T) procedures according to manufacturer specifications. Data were analyzed using FACSDiva 8.0 software.

Dissociated cells were resuspended at 1 × 10⁶ cells per ml in CMRL1066+2% FBS before the addition of HIC3-2D12/HPa3 hybridoma supernatants (at a 1:20 dilution) and biotinylated anti-CD9 (eBioscience, 13-0098-80; 1:200 dilution) and incubation at 4 °C (for 30 min). After a wash with cold CMRL1066, cells were resuspended in CMRL1066+2% FBS containing a 1:200 dilution of PE-conjugated goat anti-mouse IgM (Jackson Immunoresearch) and a 1:100 dilution of PE-Cy7-conjugated streptavidin (BD Biosciences, 557598). After another wash, cells were resuspended in CMRL1066 (Corning)+5% mouse serum (Serotec) and held on ice (for 10 min) to block the secondary antibody. A final incubation with FITC-conjugated HIC0-3C5, APC-conjugated HIC1-2B4/HPi2, APC-Cy7-conjugated HIC0-3B3/HPx1, DHIC5-4D9/HPd3, anti-CD34 (BioLegend, 343514) and APC-H7-conjugated anti-CD45 (BD Biosciences, 560178) facilitated endocrine cell subfractionation and exclusion gating of acinar (HIC0-3B3/HPx1⁺), duct (DHIC5-4D9/HPd3⁺), haematopoietic (CD45⁺) and endothelial (CD34⁺) cells. The development and characterization of several of these antibodies have been previously described12 (link). Cell doublets were excluded by pulse width measurement and propidium iodide staining was used to label dead cells for exclusion. Cells were analysed and sorted with a Cytopeia inFluxV-GS (Becton-Dickenson).