ex vivo cytotoxic potential of CD8+ T cells was measured as previously described (45 (link)). Briefly, EL-4 cells (ATCC) were pulsed with 2 µM OVA257-264 peptide (AnaSpec) or no peptide for 1hr at 37°C. Peptide pulsed cells were stained with CFSE (Life Technologies) and unpulsed cells were stained with CellTrace Violet (Life Technologies) per manufacturers instructions. CD8+ T cells were enriched by negative selection using the EasySep Mouse CD8+ T cell enrichment kit (StemCell Technologies). The number of effector cells were determined by quantification of Kb/OVA+ CD8+ T cells and the appropriate numbers were added and the mixtures were incubated for 5 hrs at 37 °C. Following incubation, cells were stained with a vital exclusion dye (Life Technologies) to exclude dead cells. Fixed cells were acquired on an LSR II flow cytometer (BD Biosciences) and analyzed using FlowJo v9.7.2 (Treestar). Specific lysis was calculated as 100 – [100 × ( % survival / average % survival in absence of effector cells)].
Vital exclusion dye
Manufactured by Thermo Fisher Scientific
Vital exclusion dye is a laboratory reagent used to assess cell viability. It functions by selectively staining non-viable cells, allowing for the identification and quantification of live and dead cells in a sample.
Automatically generated - may contain errors
Lab products found in correlation
Lsrii flow cytometer, by BD (5 mentions)
Cytofix cytoperm, by BD (2 mentions)
Murine dissociation kit, by Miltenyi Biotec (2 mentions)
Gentlemacs dissociator, by Miltenyi Biotec (2 mentions)
Facscanto, by BD (2 mentions)
Ova257 264 peptide, by AnaSpec (1 mentions)
Celltrace violet, by Thermo Fisher Scientific (1 mentions)
El4 cells, by American Type Culture Collection (1 mentions)
Easysep mouse cd8 t cell enrichment kit, by STEMCELL (1 mentions)
Carboxyfluorescein succinimidyl ester (cfse), by Thermo Fisher Scientific (1 mentions)
Perm wash, by BD (1 mentions)
Il 21r fc fusion protein, by R&D Systems (1 mentions)
Automacs rinsing solution, by Miltenyi Biotec (1 mentions)
Golgistop, by BD (1 mentions)
Golgiplug, by BD (1 mentions)
Annexin 5 staining kit, by BioLegend (1 mentions)
Foxp3 fixation permeabilization kit, by Thermo Fisher Scientific (1 mentions)
Anti ifn γ clone xmg1, by BD (1 mentions)
Brefeldin a, by BD (1 mentions)
7 protocols using vital exclusion dye
1
Cytotoxicity Assay of CD8+ T Cells
2
Characterizing SIV Env-specific CD4 T Cells
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SIV Env-specific CD4 T cells were characterized as previously described (33 (link)). Briefly, 1 × 106 to 2 × 106 splenic and iliac LN mononuclear cells were stimulated for 5 h with 1 μg/ml of an overlapping SIVmac239 Env peptide pool (NIH AIDS Reagent Program) in the presence of GolgiStop and GolgiPlug (BD Biosciences). Cells were washed three times in autoMACS rinsing solution (Miltenyi Biotec) and stained with vital exclusion dye (Life Technologies) for 10 min at 4°C. Cells were again washed and treated with Cytofix/Cytoperm (BD Biosciences) for 20 min at 4°C. All subsequent washes and stainings were performed by using BD Perm/Wash (BD Biosciences). Cells were incubated with an interleukin-21 (IL-21) receptor (IL-21R)/Fc fusion protein (R&D Systems) for 30 min at 4°C, washed three times, and stained with goat anti-human Fcγ antibody (Jackson ImmunoResearch Laboratories) for 30 min at 4°C. Cells were subsequently washed three times and stained with anti-CD4 (RM4-5), anti-CD8a (53-6.7), anti-CD44 (IM7), anti-gamma interferon (IFN-γ) (XMG1.2), and anti-IL-2 (JES6-5H4) for 30 min at 4°C. Samples were washed three additional times, fixed in 2% formaldehyde, and stored at 4°C. Samples were acquired on an LSR II flow cytometer (BD Biosciences) and analyzed by using FlowJo v9.8.3 (TreeStar).
3
Flow Cytometry of Germinal Center B Cells
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Single-cell suspensions of iliac LN mononuclear cells were blocked with TruStain fcX (anti-mouse CD16/CD32) antibodies (BioLegend) for 10 min at 4°C. Cells were washed and stained for 30 min at 4°C with anti-CD3ε (145-2C11), anti-CD19 (6D5), anti-Fas (15A7), peanut agglutinin (PNA; Vector Laboratories), anti-IgM (RMM-1), and anti-IgD (11-26c.2a). Dead cells were excluded by the use of a vital exclusion dye (Life Technologies). All antibodies were purchased from BD Biosciences, Affymetrix, or BioLegend, unless noted otherwise. Germinal center B cells were identified by flow cytometry as Fas+ PNA+ CD19+, as previously described (32 (link)). Samples were acquired on an LSR II flow cytometer (BD Biosciences) and analyzed by using FlowJo v9.8.3 (TreeStar).
4
Isolation and Phenotyping of Immune Cells
5
PBMC Isolation and SIV Gag T-cell Analysis
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Mice were bled submandibularly, and PBMCs from whole blood were isolated using Ficoll-Hypaque density centrifugation at 1,900 rpm for 20 min. Spleens were processed as previously described (24 (link)). Major histocompatibility complex class I tetramer staining was performed using the H-2Db tetramer loaded with the immunodominant AL11 peptide (AAVKNWMTQTL) as described previously (24 (link)). Biotinylated class I monomer was provided by the National Institutes of Health Tetramer Core Facility (Emory University, GA). PBMCs were surfaced stained with anti-PD-1 (clone RMP1-30), anti-CD8a (clone 53-6.7), anti-CD44 (clone IM7), and anti-KLRG1 (clone 2F1).
Splenocytes were stimulated with 1 μg/ml of an overlapping SIVmac239 Gag peptide pool. At the time of stimulation, brefeldin A (BD Biosciences) was added and samples were incubated for 5 h at 37°C. After the incubation, cells were washed and stained with the surface stain antibodies (mentioned above), permeabilized with Cytofix/Cytoperm (BD Biosciences), and stained with anti-IFN-γ (clone XMG1.2) antibodies for half an hour. Vital exclusion dye was purchased from Invitrogen. All antibodies were purchased from either BioLegend or BD Biosciences. All samples were acquired using an LSR II flow cytometer (BD Biosciences), and data were analyzed using FlowJo (version 9.6.4) software (TreeStar).
Splenocytes were stimulated with 1 μg/ml of an overlapping SIVmac239 Gag peptide pool. At the time of stimulation, brefeldin A (BD Biosciences) was added and samples were incubated for 5 h at 37°C. After the incubation, cells were washed and stained with the surface stain antibodies (mentioned above), permeabilized with Cytofix/Cytoperm (BD Biosciences), and stained with anti-IFN-γ (clone XMG1.2) antibodies for half an hour. Vital exclusion dye was purchased from Invitrogen. All antibodies were purchased from either BioLegend or BD Biosciences. All samples were acquired using an LSR II flow cytometer (BD Biosciences), and data were analyzed using FlowJo (version 9.6.4) software (TreeStar).
6
Isolating Myeloid Cells from Murine Tumors
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To obtain single cell suspensions, tumours were processed using a gentleMacs dissociator and a murine dissociation kit (Miltenyi Biotec, Surrey, UK). For staining of cells, non-specific binding was blocked with rat anti-CD16/CD32 Fc block on ice. Cells were incubated with Gr-1–FITC, CD11b–APC (eBioscience, Leicestershire, UK), washed in 1% FCS/PBS. For analysis, live cells were gated using vital dye exclusion (Invitrogen) and population phenotyped on FACs Canto (BD Bioscience) and analyzed using Flow Jo software. An example of the gating strategy employed is provided in Figure S5 .
7
Tumor Dissociation and Immune Cell Analysis
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To obtain single cell suspensions, tumours were processed using a gentleMacs dissociator and a murine dissociation kit (Miltenyi Biotec). For staining of cells, non-specific binding was blocked with rat anti-CD16/CD32 Fc block on ice. Cells were incubated with Gr-1 FITC, CD11b-APC (eBiosceince), washed in 1% FCS/PBS. For analysis, live cells were gated using vital dye exclusion (Invitrogen) and population phenotyped on FACs Canto (BD Bioscience) and analysed using Flow Jo software. An example of the gating strategy employed for selection of either CD45 -, CD45 + , or CD45 + CD11b + Gr1 + cells is provided in Supplementary Figure 2.
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