Naïve T lymphocytes were isolated and primed as mentioned above. The T cells were then harvested and stained by anti-CD4-APC and anti-IFN-γ-PE or anti-CD8-FITC and anti-IFN-γ-PE (BD Biosciences). Fluorescence profiles were measured on FACSCalibur and the data were analyzed by cell quest software (both from BD Biosciences). To deplete CD8+ and CD4+ T cells, 500 µg of either anti-CD4 (clone GK1.5, rat IgG), anti-CD8 (clone 2.43, rat IgG) or isotype controls were add to T cells 24 h before co-incubation with vaccine pulsed DCs, after 5 days of stimulation with pulsed DCs, primed T cells were collected and tested for cytotoxicity assay as mentioned above.
Anti cd8 fitc and anti ifn γ pe
Manufactured by BD
Anti-CD8-FITC and anti-IFN-γ-PE are fluorochrome-conjugated antibodies used for flow cytometry analysis. Anti-CD8-FITC binds to the CD8 surface marker, while anti-IFN-γ-PE binds to the interferon-gamma cytokine. These reagents are commonly used for the identification and quantification of CD8-positive cells and interferon-gamma-producing cells.
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2 protocols using anti cd8 fitc and anti ifn γ pe
1
T Cell Depletion and Cytotoxicity Assay
2
OVA-specific CTL Generation
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Spleen cells from the mice about 7 days after the third immunization were separated by lymphocyte separation medium and resuspended. OVA-specific CTLs were generated by stimulating spleen lymphocytes with 50 μg/mL of OVA protein for 3 days. The cells were then stained by FITC-conjugated anti-CD8 (BD Biosciences) and PE-conjugated H-2Kb/OVA257–264 complex (MBL Co., Ltd., Nagoya, Japan). For intracellular cytokine staining, spleen T cells were incubated at a density of 2 × 106 cells per mL in complete RPMI 1640 containing 10 μg/mL OVA257–264 or OVA323–339 for 24 h, and added with Golgi Stop (BD Biosciences) during the last 4–6 h. The cells were then harvested and stained by anti-CD4-APC and anti-IFN-γ-PE or anti-CD8-FITC and anti-IFN-γ-PE (BD Biosciences). Fluorescence profiles were acquired on a FACScan flow cytometer (Becton Dickinson) and analyzed using CellQuest software.
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