LIVE/DEAD Fixable Violet Dead Cell Stain Kit

Protocol tips

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	Add reconstituted fluorescent reactive dye and incubate at room temperature or on ice for 30 minutes

Protocol tips
Add reconstituted fluorescent reactive dye and incubate at room temperature or on ice for 30 minutes

Publication protocol

2x106 single-cell suspended mouse splenocytes were added per well to a U-bottom 96-well plate (ThermoFisher). Cells were stimulated for 5 hours at 37°C in 5% CO2, either in the presence of media alone (negative control), media with Cell Activation Cocktail (BioLegend) containing pre-mixed PMA and ionomycin (positive control), or media with MAYV envelope peptides (1μg/ml) spanning the length of the entire protein, where all of the samples contained a protein transport inhibitor cocktail (eBioscience, San Diego, CA, USA). Upon completed stimulation, the cells are washed with FACS buffer (PBS containing 0.1% sodium azide and 1% FBS). Cells were stained for the surface proteins using fluorochrome-conjugated antibodies per the manufacturer’s instructions (BD Biosciences, San Diego, CA, USA). The cells were washed again with FACS buffer, then fixed and permeabilized using BD Cytofix/Cytoperm (BD Biosciences) per the manufacturer’s protocol before the intracellular cytokines were stained using fluorchrome-conjugated antibodies (BD Biosciences). The following antibodies were used for surface staining: LIVE/DEAD Fixable Violet Dead Cell stain kit (Invitrogen); CD19 (V450; clone 1D3; BD Biosciences); CD4 (FITC; clone RM4-5; eBioscience); CD8α (APC-Cy7; clone 53–6.7; BD Biosciences); CD44 (A700; clone IM7; BioLegend). For intracellular staining the following antibodies were used: IFN-γ (APC; clone XMG1.2; Biolegend); TNF-α (PE; clone MP6-XT22; eBioscience); CD3ε (PerCP/Cy5.5; clone 145-2C11; Biolegend); IL-2 (PeCy7; clone JES6-SH4; eBioscience). The LSRII flow cytometer was outfitted with the following lasers and bandpass filters: (i) violet (405 nm)– 450/50, 525/50, 560/40, 585/42, 605/40, 660/40, 705/70, 780/60; (ii) blue (488 nm)– 530/30, 695/40; (iii) green (532 nm)– 575/25, 610/20, 660/20, 710/50, 780/60; and (iv) red (640nm)– 670/30, 710/50, 780/60. All data was collected using an LSRII flow cytometer (BD Biosciences) and analyzed using FlowJo software (Tree Star, Ashland, OR, USA) and SPICE v5. Boolean gating was performed using FlowJo software to examine the polyfunctionality of the T cells from vaccinated animals [21, 23, 24].

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Manufacturer protocol

Download the product protocol from Thermo Fisher Scientific for LIVE/DEAD Fixable Violet Dead Cell Stain Kit below.

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