Anti cd8 pe cy5

Mouse gp100_25–33 (mgp100, EGSRNQDWL) and human gp100_25–33 (hgp100, KVPRNQDWL) peptides were synthesized by Peptron (Daejeon, Korea). CD8 microbeads were purchased from Miltenyi Biotec (Auburn, AL, USA). All antibodies used for flow cytometry were purchased from BD Bioscience, including anti-Thy1.1-FITC, anti-CD3-FITC, anti-B220-FITC, anti-CD62L-FITC, anti-Ly-6C-FITC, anti-CD19-PE, anti-CD8-PE, anti-CD44-PE, anti-CD8-PE-Cy5, anti-Thy1.1-PE-Cy5, anti-CD11b-PE-Cy5, anti-CD4-APC, anti-CD8-APC, and anti-CD45-APC. Recombinant human IL-2 (Proleukin) was purchased from Novartis, and the Fc fusion protein of human IL-7 (IL7-Fc), which consist of the extracellular domain of human IL-7 (aa 26–177) fused to the N-terminus of the Fc portion of a mutant human IgG1, was obtained from AdipoGen (Seoul, Korea). The CellTrace CFSE Cell Proliferation Kit was purchased from Invitrogen (Carlsbad, CA, USA).

For flow cytometry, splenocytes were stimulated with 0.7 μl/ml of 50 ng/ml PMA and 500 ng/ml IO, and then 1μg/ml of berefeldinA was added to retain the cytokine in the cytoplasm. After incubation for 4 h at 37°C with 5% CO₂, these cells were stained for CD4 or CD8 surface markers using anti-CD4-PEcy5 or anti-CD8-PEcy5 antibodies (all from BD Biosciences) in separate tubes and incubated for 30 min at 4°C in the dark,. For IFN-γ and IL-4 intracellular staining after fixation and permeabilization using a Cytofix/Cytoperm^™ Plus Fixation/Permeabilization Kit with BD Golgi PlugTM (cat no.555028), cells were stained with anti-IFN-γ-FITC and anti-IL-4-PE antibodies and incubated for 30 min at 4°C in the dark. Stained cells were analyzed using FACSCalibur^™ (BD Biosciences).

CD8⁺ T cells were enriched from 2C splenocytes by negative selection using a CD8⁺ T cell isolation kit (Miltenyi Biotec, Auburn, CA) according to the manufacture's instruction. After the negative isolation the CD8⁺ T cells were stained with the anti-2C TCR-FITC mAb (clone 1B2), anti-CD3-PE (BD-Pharmingen) and anti-CD8-PECy5 (BD-Pharmingen) to evaluate the purity of the population. The isolated 2C CD8⁺ T cells (3 × 10⁶) were adoptively transferred by i.v. injection in naïve B6 mice.

Responses of NK cells and T cells were assessed as described previously.15 (link) Briefly, cells were stained with fluorophore-labelled monoclonal antibodies to cell surface molecules, fixed, permeabilized and stained for intracellular molecules using a Cytofix/Cytoperm kit (BD Biosciences). Cells were analysed by flow cytometry on an LSR II (BD Biosciences). Samples with fewer than 100 NK cells in each subset were excluded. The following reagents were used: anti-CD56-phycoerythrin (PE) -Cy7, anti-CD16-allophycocyanin (APC) -H7, anti-CD4-Pacific Blue, anti-IFN-γ-e780, anti-IFN-γ-APC, anti-CD3-V500 and anti-CD69-phycoerythrin-cyanine5 (PE-Cy5) (all BD); anti-CD8-PE-Cy5, anti-CD25-PE, anti-IL-18Rα-PE, anti-CD62L-PE-Cy5, anti-CD57-e450 and anti-IL-2-APC (all e-Biosciences/Affimetrix, Hatfield, UK). Anti-IL-12Rβ2 monoclonal antibody was obtained from R&D Systems (Oxford, UK) and conjugated to PE-Cy5 using an Easylink PE/Cy5® Conjugation Kit (Abcam, Cambridge, UK).

Whole blood was permeabilized and fixed using cytofix/cytoperm (BD Pharmingen, San Jose, CA, USA) according to manufacturer’s protocol followed by staining for 20 min at room temperature in the dark with cocktail of antibodies including anti-CD4-APC, anti-CD25-FITC, anti-FOXP3-PE, anti-PD1-PeCy7, anti-IL10-APC, anti-CD127-APC, anti-CD8PeCy5, anti-NOTCH1 PE, and anti TGF-β APC (BD Pharmingen, CA, USA). After staining, RBCs were lysed using BD FACS lysing solution (BD Pharmingen, San Jose, CA, USA) as per manufacturer’s instructions.
Anti-Notch1 PE antibody (clone mN1A) was procured from eBiosciences, CA, USA; mN1A antibody reacts with the intracellular domain of human Notch1. The mN1A antibody has a low affinity for the full-length (unprocessed or heterodimeric cell surface) forms of Notch1. Therefore, Notch1 expression was considered intracellular not surface expression.
More than 50,000 cells were acquired for flow-cytometric analyses on BD FACS Caliber and the results were analyzed using the TreeStar Flow-Jo software version 8.8.7.