Live dead fixable violet dead cell staining kit

Cell viability was determined using the CellTiter^® 96 AQueous Cell Proliferation Assay Kit (Promega) according to manufacturer’s instructions. In brief, 20 μl of reagent was added to each well of a 96-well plate containing OC and 200 μl of culture medium, incubated for a further 2 hrs, and absorbance at 490 nm was recorded using an ELISA plate reader. In certain experiments viability was also quantified by flow cytometry using the LIVE/DEAD® Fixable Violet Dead Cell staining kit (Invitrogen).

Spleen and lung lymphocytes were stained with PE-conjugated MHC class I H-2k^b dextramers loaded with OVA_257–264-peptide (SIINFEKL) (Immudex), for the detection of OVA-specific CD8 T cells, respectively, followed by anti-CD8α, CD44, and CD4. Antibodies were purchased from BD Biosciences, eBioscience, or BioLegend. For detection of adoptively transferred OT-I CD8⁺ T cells and gut-homing receptors, MLN cells were stained with anti-CD8α, CD45.1, CD44, α4β7, and CCR9. For intracellular cytokine staining, spleen and lung mononuclear cell suspensions were incubated with 2.5 µg/ml of MHC class I restricted peptides (OVA_257–264) (Genscript) for 5–6 h at 37°C in complete RPMI in the presence of 1 µg/ml GolgiPlug (BD Biosciences). Cells were stained as before with anti-CD8α, CD44, CD4, IFN-γ, and TNF-α. Intracellular cytokine staining of IFN-γ and TNF-α were performed after fixation/permeabilization (BD Cytofix/Cytoperm, BD Biosciences). For live/dead discrimination cells were stained before fixation (LIVE/DEAD fixable violet dead cell staining kit, Life Technologies). For analysis, all cells were gated on live CD8⁺ T lymphocytes following live/dead staining. All cells were acquired using a digital flow cytometer (LSR II, BD Biosciences) and data were analyzed with FlowJo software (Tree Star).