Perforin biotinylated pf 344

The following antibodies were used for culturing or surface and intracellular cytokine staining (ICS) for flow cytometry: CD28 (CD28.2, BD), CD49d (9F10, BD), CD3-PB (SP34-2, BD), CD4-BV510 (L200, BD), CD8-PB (RPA-T8, BD), IFN-γ-APC (4S.B3, BD), IFN-γ Brilliant Violet 711 (4S.B3, Biolegend), TNF-α-PE (MAb11, BD), TNF-α-PE-Cy7 (MAb11, BD), IL-17-PE (eBio64CAP17, eBioscience), IL-22-biotinylated (anti-human IL-22, RD), Streptavidin-Pacific blue (Invitrogen), Perforin-biotinylated (Pf-344, Mabtech), Caspase 3-AF647 (C92-605,BD), and anti-Vγ2-FITC (7A5, Pierce).
After staining, the cells were fixed and subjected to flow cytometry. Lymphocytes were gated based on forward- and side-scatters, and at least 40,000 gated events were analyzed using Summit Data Acquisition and Analysis Software (DakoCytomation).

The following Abs were used for culture or surface and intracellular cytokine staining for flow cytometry: CD28 (CD28.2, BD), CD49d (9F10, BD), CD3-Pacific blue (SP34-2,BD), CD4-BV510 (L200, BD), CD8- Pacific blue (RPA-T8, BD), IFN-γ-APC (4S.B3, BD), IFN-γ Brilliant Violet 711 (4S.B3, Biolegend), TNF-α-PE (Mab11, BD), TNF-α-PE-Cy7 (Mab11, BD), IL-17-PE (eBio64CAP17, eBioscience), IL-22-biotinylated (anti-human IL-22, RD), Streptavidin-Pacific blue (invitrogen), Perforin-biotinylated (Pf-344, Mabtech), Caspase 3-AF647 (C92-605,BD), anti-Vγ2-FITC (7A5, Pierce).
After staining, cells were fixed and subjected to analysis on flow cytometer of BD LSRFortessa^TM Cell Analyzer. Lymphocytes were gated based on forward- and side-scatters, and at least 40,000 gated events were analyzed using Summit Data Acquisition and Analysis Software (Dako Cytomation).