Biotinylated anti h2kd

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).

Mouse islet cells were stained with anti-CD45 and biotinylated anti-H2Kd (BD Pharmingen) followed by streptavidin-allophycocyanin (BioLegend). β‐cells were identified by autofluorescence (23 (link)). Dead cells were excluded using propidium iodide. The immune cells infiltrating islets and spleens were stained with anti-CD45, anti-CD3, anti-CD4, anti-CD8a, anti-CD44, anti-CD62L, anti-KLRG-1, anti-CD11b, anti-Ly6G (all BioLegend), anti-BrdU, anti-pSTAT3, and anti-pSTAT5 (all BD Bioscience). Data were collected on the FACS Fortessa cell analyzer (BD Bioscience, San Jose, CA, USA) and were analyzed using FlowJo Software version 10 (TreeStar, Inc, Ashland, OR, USA).