Cytokine capture assays and flow cytometry were performed using previously described methods.8 Briefly, T cells were cocultured with autologous DCs (5:1 ratio), pulsed with peptides, polyclonally stimulated with staphylococcal enterotoxin B (SEB) as a positive control, or maintained in normal medium as a negative control. Tumor necrosis factor alpha (TNF-α) and IFN-γ secretion detection kits (Miltenyi Biotec GmbH) were used to label secreted cytokines as described previously.21 The following reagents were used: yellow dead cell stain (Life Technologies GmbH; L34959; 1:1,000), anti-human CD4-PerCP-Cy5.5 (clone RPA T4, BD), anti-CD8-V450 (RPA T8, BD), anti-CD45RA-APC-H7 (clone H100, BD), and anti-CD62L-PE-Cy7 (clone DREG56, eBioscience) antibodies. Data were collected using the FACSCanto instrument with FACSDiva software (BD, San Jose, CA). We used the same gating strategy as reported previously.8 The data were analyzed and visualized using FlowJo 10.4 software (FlowJo, Ashland, OR) and GraphPad Prism version 7 (GraphPad, San Diego, CA).
Anti cd8 v450 rpa t8
Manufactured by BD
Anti-CD8-V450 (RPA T8) is a monoclonal antibody conjugated with the V450 fluorochrome. It is designed for the detection and analysis of CD8-positive cells in flow cytometry applications.
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Lab products found in correlation
2 protocols using anti cd8 v450 rpa t8
1
Cytokine Profiling of T Cell Responses
2
Multiparametric Flow Cytometry Analysis
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After stimulation, 2 mM EDTA (final concentration) was added to each well for 10 min at room temperature (RT). A LIVE/DEAD Fixable Dead Cell Stain (blue fluorescent reactive dye; Invitrogen) was used to identify and exclude non-viable cells from the analysis. Cells were fixed (BD Lyse Solution) and permeabilized (BD Perm 2), according to manufacturer's instructions. Surface and intracellular staining were done simultaneously for 30 min at RT with the following pre-titered antibody panel: anti–CD3 PerCP Cy5.5 (UCHT1; BD), anti–CD8 V450 (RPA-T8; BD), anti–CD4 V500 (RPA-T4; BD), anti–IFNγ PE-Cy7 (B27; BD), anti–IL-2 PE (5344.111; BD), anti–TNFα FITC (6401.1111;BD), and anti–MIP-1β APC (D21-1351; BD). Cells were acquired using an LSRII flow cytometer (BD) within 24 hours and ≥200,000 total events were collected for each sample unless otherwise specified.
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