Pe cy5 conjugated streptavidin

Staining was performed on peritoneal exudate cells using the following anti-mouse antibodies and flurorochrome conjugates: anti-CD4 with Biotin (Miltenyi Biotec), anti-CD25 with PE (eBioscience) and anti-Foxp3 with APC (Miltenyi Biotec). Secondary staining for biotinylated CD4 was done with either FITC- or Pe-Cy5-conjugated streptavidin (BD Biosciences). As isotype control of activated/regulatory T-cells, mouse IgG1 K isotype (APC, Miltenyi Biotec) and Rat IgG1 K Isotype (PE, eBioscience) were used. The staining procedure was executed as previously described (6 (link)) and results acquired on a cytometer (FACSCalibur^TM, Beckton Dickinson) and analyzed on FlowJo software (Tree Star, United States).

SCID mice were implanted SQ with patient tumor xenografts as previously described [14 (link), 15 (link)]. When tumors reached 100 mm³, mice were treated with 200 μg of drozitumab by intraperitoneal (IP) injection. To assess the degree of in vivo apoptosis, tumors were excised approximately 5 h after treatment with drozitumab and dissociated with a Miltenyi gentleMACs Tissue Dissociator (Miltenyi, San Diego, CA) into a single cell suspension in order to minimize cellular damage. The tumor cells were then stained with anti-CD24, anti-CD44, anti-CD326/ESA, and biotinylated anti-H2K^D BD Biosciences, Franklin Lakes, NJ. A PE-Cy5 conjugated streptavidin (BD Biosciences, Franklin Lakes, NJ) secondary was used to label the biotinylated anti-H2K^D. To determine the level of apoptosis, cells were permeabilized with BD Cytoperm/Cytofix (BD Bioscience), stained intracellularly for cleaved capase-3 (BD Bioscience). Staining was assessed on an LSR II flow cytometer (BD Biosciences), and data was analyzed using FCS Express software (DE Novo).