Fixable viability dye efluor 506 or 780

Manufactured by Thermo Fisher Scientific

Sourced in United States

The Fixable Viability Dye eFluor 506 or 780 is a fluorescent dye used for distinguishing live and dead cells in flow cytometry applications. It binds to proteins on the cell surface, allowing for the identification of dead cells. The dye emits fluorescence in the 506 or 780 nanometer range, respectively, which can be detected by flow cytometers.

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4 protocols using fixable viability dye efluor 506 or 780

Multiparameter Flow Cytometry Staining

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For samples not sorted, cells were washed with PBS and stained in FACS buffer (2% FBS, 2 mM EDTA, and 0.001% NaN₃). All gating strategies are represented in Supplementary Fig. 9. Cells were first stained for H-2K^b/SIY-pentamer (PE; ProImmune) for 10 min at room temperature at a 1:20 dilution, followed by staining with remaining antibodies for 20 min on ice. Antibodies against the following molecules were used: CD3ε (clone 17A2, BioLegend, 100216), Thy1.2 (clone 30-H12, BioLegend, 105320), CD45.2 (clone 104, BioLegend, 109806), CD8α (clone 53-6.7, BioLegend, 100747), CD4 (clone RM4-5, BioLegend, 100547), PD-L1 (clone 10 F.9G2, BioLegend, 124312), CD19 (clone 6D5, BioLegend, 115545), I-A/I-E (clone M5/114.15.2, BioLegend, 107630), and H-2K^b (clone AF6-88.5, BioLegend, 742862). All antibodies were used at a 1:100 dilution. Fixable Viability Dye eFluor 506 or 780 (eBioscience) was used for live/dead discrimination and was used at a 1:200 dilution. All flow cytometric analysis was conducted on either an LSRFortessa or X20 (BD) and analyzed using FlowJo software (Tree Star).

Williams J.B., Li S., Higgs E.F., Cabanov A., Wang X., Huang H, & Gajewski T.F. (2020). Tumor heterogeneity and clonal cooperation influence the immune selection of IFN-γ-signaling mutant cancer cells. Nature Communications, 11, 602.

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Characterization of Decidual Immune Cells

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All antibodies used for characterization of decidual stromal cells, and for identification of decidual T cells, NK cells, NK-T cells, B cells and pDCs are listed in Table 1. To identify living leukocytes, cells were stained with Fixable Viability Dye (eFluor 506 or 780, eBioscience, San Diego, USA). For experiments analyzing intracellular IFN-γ and IFN-α production, isolated decidual mononuclear cells (10⁶/ml) were cultured overnight with or without poly(I:C) together with IL-12 (10 μg/ml and 10 ng/ml (Nordic Biosite, Stockholm, Sweden), respectively). Brefeldin A (5 μg/ml, BD Biosciences, New Jersey, USA) was added for the last 3 hours. After surface staining cells were fixed and permeabilized using Cytofix/Cytoperm™ kit (BD Biosciences). Antibodies used for detection of IFN-γ and IFN-α are listed in Table 1. Samples were acquired in a FACSVerse or FACSCanto II (BD Biosciences) equipped with FACSSuite or FACSDiva software and analyzed with FlowJo software (TreeStar, Ashland, USA).

Lundell A.C., Nordström I., Andersson K., Lundqvist C., Telemo E., Nava S., Kaipe H, & Rudin A. (2017). IFN type I and II induce BAFF secretion from human decidual stromal cells. Scientific Reports, 7, 39904.

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Quantification of Plasma Cell Differentiation

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In contrast to the other effector cell assays, B cells were co-cultured directly with freshly isolated DCs. Autologous B cells were incubated with or without DCs at a 10:1 ratio (B cell:DC) in the presence of different stimuli, for 7 d at 37°C. DCs were CD1c⁺ DCs, pDCs or a combination of both at a 1:1 ratio, with each well containing equal total DC numbers. After 7 d, B cells were stained with CD20-APC (eBioscience, 17-0209-42) and CD38-PE (ImmunoTools, 21270384) or CD38-PerCP-Cy5.5 (BD Biosciences, 551400) and a viability dye (Fixable Viability Dye eFluor 780 or 506; eBioscience, 65-0865-18 or 65-0866-18) and measured on a FACSVerse. Plasma cells were defined as CD38^highCD20^low expressing cells.

van Beek J.J., Gorris M.A., Sköld A.E., Hatipoglu I., Van Acker H.H., Smits E.L., de Vries I.J, & Bakdash G. (2016). Human blood myeloid and plasmacytoid dendritic cells cross activate each other and synergize in inducing NK cell cytotoxicity. Oncoimmunology, 5(10), e1227902.

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Flow Cytometry Analysis of Tumor Cell Lines

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GD2 expression on tumor cell lines was performed using the anti-GD2 (clone 14g2a; BD Pharmingen) and compared to isotype controls. T-cell transduction was measured using the 14g2a anti-idiotype, clone 1A7 (Biological Research Branch of NCI) conjugated to Dylight 650 or 488 (Pierce Protein Biology Products). T-cell phenotypes were identified using the following antibodies to human CD4 (clone OKT4; eBioscience), human CD8 (clone RPA-T8; eBioscience), human CD45 (clone H130; eBioscience), human PD-1 (clone eBioJ105, eBioscience), human TIM-3 (clone F35-2E2, eBioscience), and human LAG-3 (clone 3DS223H, eBioscience). MDSCs were evaluated using antibodies to Ly6C (clone HK1.4; eBioscience), Ly6G (clone 1A8; eBioscience), CD11b (clone M1/70; eBioscience). All antibodies were used per manufacturers recommendations. Live/dead cells were distinguished with Fixable Viability Dye eFluor 780 or 506 (eBioscience). Flow cytometry was done using FACS Fortessa with FACS Diva software (BD Biosciences) and analyzed by FlowJo 9 software (Treestar).

Long A.H., Highfill S.L., Cui Y., Smith J.P., Walker A.J., Ramakrishna S., El-Etriby R., Galli S., Tsokos M.G., Orentas R.J, & Mackall C.L. (2016). Reduction of MDSCs with all-trans retinoic acid improves CAR therapy efficacy for sarcomas. Cancer immunology research, 4(10), 869-880.

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