Förster, Hannover, Germany), anti-CD4-APC, anti-CD25-PE (both acquired from Serotec, Oxford, GB), and anti-Foxp3-FITC (eBiosciences, San Diego, USA). DCs were identified by anti-MHC class II-PE, anti CD11c-APC and CD103-FITC (all acquired from BD Biosciences, Heidelberg, Germany). All FACS analyses were performed on a FACSCanto (BD Biosciences). Isotype-matched mAb served as controls. Immunoglobulin (Ig) isotyping was performed using the mouse immunoglobulin isotyping ELISA Kit (BD Biosciences, San Diego, USA). Serum

samples were diluted at a concentration of 1:2000 to achieve optical density Vismodegib in a range of 0.5–1.2. Furthermore, the concentration of OVA-specific Ig in the serum was analyzed in the ELISA. Therefore, the plates were coated with 0.5 μg/mL OVA (Grade VI; Sigma-Aldrich) in PBS overnight at 4°C. After washing, the

plates were blocked and samples were added to a concentration of 1:10 to 1:500 and incubated 120 min at 37°C. After washing, detection Abs (biotinylated anti-IgG1; anti-IgG2a, anti-IgG2b, anti-IgG3, anti-IgA, anti-IgM; BD Biosciences) were added and later detected with horseradish peroxidase (HRP, BD Biosciences), tetramethylbenzidene (TMB, BD Biosciences) and hydrogen peroxide (1:1) as the substrate. The reaction was stopped with 2NH2SO4 (Merck, Darmstadt, Germany). The optical density was analyzed in an ELISA-reader (Bio-TEK Instruments GmbH, Bad Friedrichshall, Germany). Calculations, statistical analysis and graphs were performed on Graphpad Prism 4.0 (Graphpad Software, compound screening assay San Diego, USA). The comments of Astrid Westendorf have been a great help. The authors also wish to thank Melanie Bornemann for excellent technical assistance, Tim Worbs for advice on DTH reaction and Sheila Fryk for correction of the English. The Progesterone work was supported by the Deutsche Forschungsgemeinschaft (SFB621/A10).

The work was supported by the German Research Foundation (SFB621/A10). Conflict of interest: The authors declare no financial or commercial conflict of interest. Detailed facts of importance to specialist readers are published as ”Supporting Information”. Such documents are peer-reviewed, but not copy-edited or typeset. They are made available as submitted by the authors. “
“This unit describes two different protocols for the measurement of tumor cytolysis by macrophages. Traditionally, cytotoxicity assays have relied on the use of radioactive isotopes. In Basic Protocol 1, cytotoxic activity is measured by the release into the culture supernatant of a radioisotope that had been incorporated by the target cell and is released upon cell death. This poses a problem for some cell lines in which spontaneous isotope release occurs in the absence of effector cell cytotoxicity. In Basic Protocol 2, a nonradioactive approach is used to measure cytolysis that relies on the fluorescence staining of tumor cells with cell-death markers.