Fluorochrome conjugated monoclonal antibodies mabs

For intracellular cytokine staining, cells were stimulated at 37°C for 5 hours with a Leukocyte Activation Cocktail (BD Pharmingen). Cells were then stained with surface markers, fixed, and permeabilized with IntraPre Reagent (Beckman Coulter), and finally stained with intracellular markers. Data were acquired on FACSVantage SE and analyzed with CellQuest software. Fluorochrome-conjugated monoclonal antibodies (mAbs) against IL-23A, CD3 served as marker for T cells and CD14 serve as markers for monocyte and macrophage were purchased from BD Pharmingen.

Fluorochrome-conjugated monoclonal antibodies (mAbs) specific for human antigens were obtained, unless stated otherwise, from BD Pharmingen (San Jose, CA, USA), BioLegend (San Diego, CA, USA), or eBioscience (San Diego, CA, USA). The following anti-human mAbs (clone) were used: FasL (NOK-1), Fas (DX2), CD19 (H1B19), CD5 (L17F12), CD40 (5C3), CD86 (IT2.2), CD20 (2H7), CD27 (LG.7F9), CD38 (HIT2), CD22 450 (S-HCL-1), CD10 (HI10a), CD1d (51.1), IL-10 (JES3-9D7), active caspase 3 (C92-605), IL-17 (eBio64DEC17), and IFN-γ (B27). Surface expression of human FasL was analyzed by using biotin-conjugated NOK-1 mAb. Previously described (25 (link), 26 (link)) humanized anti-human FasL (RNOK203) was provided by Dr. Hideo Yagita of Juntendo University and Dr. Toshihiro Maeda of The Chemo-Sero Therapeutic Research Institute (Kaketsuken) in Japan.