Goat anti mouse pe conjugate

Paraformaldehyde was purchased from Affymetrix (Santa Clara, CA). Anti-human TACI antibody (clone 165604) and the respective isotype control were from R&D Systems (Minneapolis, MN). CD41a-PeCy5, CD61-FITC and CD62P-FITC were from BD Pharmingen (San Diego, CA). The goat anti-mouse PE conjugate was from Dako (Glostrup, Denmark). Bicoll Separating Solution was purchased from Biochrom AG (Berlin, Germany). Recombinant human BAFF (rhBAFF) and recombinant human APRIL (rhAPRIL) was from PeproTech (Rocky Hill, NJ, USA). Thrombin Receptor Activator Peptide 6 (TRAP-6), collagen and ADP was purchased from SigmaAldrich (St. Louis, MO). Citrate buffer contained 10 mM sodium citrate, 150 mM NaCl, 1 mM EDTA, 1% dextrose, pH 7.4. GI254023 was from Tocris (Bristol, UK) and Batimastat was from Calbiochem (Darmstadt, Germany).

For analysis of B7-H3 surface expression and B7-H3xCD3 binding, cells were stained with a parental murine B7-H3 antibody (10 µg/mL) carrying the same B7-H3 binding clone as our construct (7C4), B-H3xCD3 or the corresponding isotype controls followed by a goat anti-mouse-PE conjugate (Dako, Glostrup, Denmark) or a donkey anti-human-PE conjugate (Jackson ImmunoResearch, West Grove, USA), respectively. T cell activation, degranulation and proliferation were determined using CD69-PE, CD107a-PE (BD Pharmingen) as well as CD4-APC, CD8-FITC, CD62L-PB and CD45ro-PeCy7 (BioLegend, San Diego, CA) fluorescence conjugates. For flow cytometric analysis of target cell lysis, tumor cells were loaded with 2.5 µM CellTrace™ Violet (Thermo Fisher Scientific, Waltham, MA) and cultured with monocyte-depleted PBMC (E:T 4:1) in the presence or absence of B7-H3xCD3 or MOPCxCD3 (1 nM each). Standard calibration beads (Sigma-Aldrich, St. Louis, MO) were used to ensure analysis of equal assay volumes and to account for the number of target cells that had vanished from the culture. 7AAD (Biolegend) was used for live- and dead-cell discrimination. Measurements were performed using a FACS Canto II or FACS Fortessa (BD Biosciences, San Diego, CA) and data was analyzed using the software FlowJo (FlowJo LCC, Ashland, OR).

Paraformaldehyde was from Affymetrix (Santa Clara, CA, USA). Anti-OX40L antibodies were from Ancell Corporation (Bayport, MN, USA). Anti-human TACI, anti-human GITRL antibody and the respective isotype control were from R&D Systems (Minneapolis, MN). CD41a-PeCy5, and CD62P-FITC were from BD Pharmingen, CD3-APC/Fire, CD69-PE and CD25-FITC and CD56-PECy7 were obtained from BioLegend. The goat anti-mouse PE conjugate was from Dako (Glostrup, Denmark). Dead cells were excluded using Fixable Aqua (Invitrogen, Carlsbad, CA, USA) after extracellular staining according to the manufacturer’s instructions. Bicoll Separating Solution was purchased from Biochrom AG (Berlin, Germany).