12367 Version Date: 10/24/2017 Version: 09 F ACSCalibur (Beckton Dickinson) and analyzed them using FlowJo software (Tree Star). This method has been previously published by Chalmin et al.11 9.3.2 Determination of serum cytokine and chemokine levels S erum levels of proinflammatory cytokines and chemokines induced by radiation therapy will be ascertained through the Luminex Protein Assay (Invitrogen) as per manufacturer instructions. 9.3.3 Detection of circulating mtDNA and other TLR9 ligands L evels of TLR9 ligands (e.g. mtDNAs), will be assessed using quantitative qPCR as described by Zhang et al.(Blood 2013). Briefly, DNA will be isolated from plasma samples using QIAamp DNA Mini kit (Qiagen). The presence of human mitochondrial genes, such as cytochrome B, cytochrome C oxidase subunit III and NADH dehydrogenase, will be detected by qPCR using specific pairs of primers on CFX96 thermocycler (Biorad). Page 33 of 50 IRB Protocol No. 12367 Version Date: 10/24/2017 Version: 09 10.0 Study Calendar Table 1 Schedule of Events (time scale in weeks) ARM B # Week f Pre-Study * 1 2 3 4 5 6 7 8 9 10 11 12 b Off Study Visiti Radiation therapy # (Arm B only) X X Sipuleucel -T collection X X X Sipuleucel -T infusion X X X Informed consent X Demographics X Medical history X X Physical exam X X X X X X Performance Status X X X X X X CBC w/diff, plts X X X X X X X Xe Serum chemistry a X X X X X Xe PSA X X X Xe Testosterone X X EKG (as indicated) X CXR (if no CT chest done) X Xc Xc Radiologic evaluation(bone scan, CT (Chestg) Abd/pelvis g X Xc Xc Tumor measurements X Xc Xc Adverse event evaluation X X X Xc Xc Immune Assays X X Xd Analysis of PBMCsh X X # Patients on ARM A will not undergo radiation therapy, otherwise they will follow the same schedule as ARM B beginning on wee k 3 * Prestudy tests should be perform ed within 28 days of registration a. Albumin, alkaline phosphatase, total bilirubin, bicarbonate, BUN, calcium, chloride, creatinine, glucose, LDH, phosphorus, potassium, total protein, SGOT[AST], SGPT[ALT], sodium.