Human macsplex cytokine 12 kit

Supernatants from cultured cells from the cross-presentation assay were monitored using the human MACS-Plex Cytokine 12 Kit purchased from Miltenyi Biotec (cat. #130-099-169). Acquisitions and analyses were performed on CytoFLEX S purchased from Beckman Coulter (cat. #B75442)/FACSAria Fusion purchased from BD Biosciences and FlowJo Software from Treestar, respectively. Supernatants from cultured cells from the peptide-based assay were monitored using ELISA tests purchased from BioLegend: ELISA MAX Deluxe Set Human IFNγ (cat. #430104), ELISA MAX Deluxe Set Human IL17 (cat. #433914), and ELISA MAX Deluxe Set Human IL9 (cat. #434705).

Cytokines secreted in the colon were analyzed with the LegendPlex multiplex assay (Biolegend) using a fluorescence-encoded bead staining procedure, and the data were acquired on a MACSQuant Analyzer 10. Mouse cytokines (IL1α, IL1β, IL10, IL12, IL17a, IL23, IL27, IFN-β, IFN-γ, TNF-α, and GM-CSF) and key targets involved in hematopoietic stem-cell differentiation and lineage-specific markers (IL34, IL5, M-CSF, TPO, IL6, GM-CSF, IL15, TGF-β1, IL3, LIF, SCF, EPO, and CXCL12) were simultaneously quantified. For PBMCs, purified CD4+ cells and macrophages, the levels of IL6, IL17a, TNF-α, and CCL2 were quantified by means of a staining procedure according to the Human MACSplex cytokine 12 KIT (Miltenyi Biotec) or the LegendPlex inflammation panel (Biolegend), and the data were acquired on a MACSQuant Analyzer 10. Cytokine profile analysis was performed with MACSQuantify (2.11) software (Miltenyi Biotec) or Quognit software (Biolegend).

Cytolytic activity was assessed by co-culturing 1E4 CAR-modified or non-CAR-modified control T cells (mock) and GFP-labeled target cells at an effector-to-target ratio of 1:1 for 24 hours before analyzing the frequency of residual viable target cells by flow cytometry. Cytokine production was analyzed from the same co-culture by harvesting 100 µL supernatant from the 24 hours co-culture and quantification using the human MACSPlex Cytokine 12 Kit (Miltenyi Biotec) according to the manufacturer’s instructions.
All in vivo studies were approved by the Institutional Animal Care and Use Committee of the University of Würzburg. In brief, NSG mice (NOD.Cg-Prkdc^scidIl2rg^tm1Wjl/SzJ, Charles River) were inoculated with 1E6 firefly luciferase expressing Raji tumor cells by intravenous (i.v.) injection, and 7 days later treated with 4.7E6 T cells from the final cell product (from run #3, containing 2E6 CAR T cells) or 4.7E6 mock T cells (also i.v.). Serial bioluminescence imaging on an in vivo imaging system Lumina imager (Perkin Elmer) was performed to assess tumor burden and distribution.