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Ebioscience fixable viability dye efluor 660

Manufactured by Thermo Fisher Scientific
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The EBioscience Fixable Viability Dye (FVD) eFluor 660 is a fluorescent dye used to identify and exclude dead or dying cells in flow cytometry analysis. It binds to dead cells, allowing them to be gated out during data acquisition and analysis.

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Lab products found in correlation

Accutase, by Merck Group (2 mentions) Polystyrene facs tubes, by Sarstedt (1 mentions) Ebioscience permeabilization buffer, by Thermo Fisher Scientific (1 mentions) Alexa fluor 488 anti cytochrome c antibody, by BioLegend (1 mentions) Facscalibur flow cytometer, by BD (1 mentions) Mouse igg1 isotype control, by Thermo Fisher Scientific (1 mentions) Cd11a pe, by BD (1 mentions) Cd11c bv650, by BD (1 mentions) Cd18 apc, by BD (1 mentions) Cd11b percp, by BD (1 mentions)

Spelling variants of this product

Fixable Viability Dye (414 citations) Viability dye (78 citations) EFluor 660 (35 citations) Fixable Viability Dye eFluor 660 (28 citations)

4 protocols using ebioscience fixable viability dye efluor 660

1

Staining and Flow Cytometry of THP-1 Macrophages

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THP-1 macrophages were stained with eBioscience Fixable Viability Dye (FVD) eFluor 660 (Thermo Fisher), detached with Accutase (Sigma-Aldrich), and transferred to polystyrene FACS tubes (Sarstedt). Single-cell suspensions were washed with MACS buffer (0.5% bovine serum albumin, 2mM EDTA in PBS, ph7.2) at 290 x g for 5 minutes at 4°C, and in most cases, fixed in 4% PFA solution overnight at 4°C. The following day, samples were washed with and resuspended in MACS buffer. Data were acquired in LSRII or Fortessa flow cytometers (BD Biosciences) and analyzed in FlowJo 10 (TreeStar), gating on single-cell events.
Pagán A.J., Lee L.J., Edwards-Hicks J., Moens C.B., Tobin D.M., Busch-Nentwich E.M., Pearce E.L, & Ramakrishnan L. (2022). mTOR-regulated mitochondrial metabolism limits mycobacterium-induced cytotoxicity. Cell, 185(20), 3720-3738.e13.
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2

Cytochrome c Release Assay for Infected Cells

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The cytochrome c release assay was adapted from Lienard et al. (2020) (link). 7 hours after infection with tdTomato-expressing Mm, cells were stained with eBioscience Fixable Viability Dye (FVD) eFluor 660 (Thermo Fisher), detached with Accutase (Sigma-Aldrich), transferred to polystyrene FACS tubes (Sarstedt), and washed first with ice-cold MACS buffer (0.5% bovine serum albumin, 2mM EDTA in PBS, ph7.2) and then ice-cold PBS by centrifugation at 290 x g for 5 minutes at 4°C. Cells were incubated in ice-cold permeabilization buffer (50 μg/mL digitonin, 100 mM KCl) for 5 minutes and immediately fixed in 4% PFA solution to stop the permeabilization reaction. Cells were then centrifuged as above and fixed overnight in 4% PFA solution at 4°C. Samples were subsequently permeabilized with eBioscience Permeabilization Buffer (Thermo Fisher) and stained with AlexaFluor 488 anti-cytochrome c antibody (BioLegend) for 2 hours at room temperature. Samples were analyzed by flow cytometry by gating on the viable, infected (FVD eFluor 660- tdTomato+) single-cell events.
Pagán A.J., Lee L.J., Edwards-Hicks J., Moens C.B., Tobin D.M., Busch-Nentwich E.M., Pearce E.L, & Ramakrishnan L. (2022). mTOR-regulated mitochondrial metabolism limits mycobacterium-induced cytotoxicity. Cell, 185(20), 3720-3738.e13.
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3

Quantifying ICAM-1 Expression on MCs

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MCs (0.1 × 106/100 µL) were incubated with fluorescently labelled antibodies, FITC-conjugated mouse anti-human ICAM1 (clone RR1/1), subclass IgG1) or mouse IgG1 isotype control (eBiosciences, Cheshire, UK) for 30 min with the addition of eBioscience™ Fixable Viability Dye eFluor™ 660 (Thermo Fisher Scientific, Paisley, UK) on ice prior to resuspending in 300 µL FACS buffer. Flow cytometry was performed using a BD FACSCalibur flow cytometer (BD Biosciences, Oxford, UK) and data analysed using FlowJo software (version 7.6.5, Oregon, BD, USA).
Akoto C., Willis A., Banas C.F., Bell J.A., Bryant D., Blume C., Davies D.E, & Swindle E.J. (2022). IL-33 Induces an Antiviral Signature in Mast Cells but Enhances Their Permissiveness for Human Rhinovirus Infection. Viruses, 14(11), 2430.
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4

Integrin Subunit Expression Quantification

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All antibodies and reagents were purchased from BioLegend (San Diego, CA) except when indicated otherwise. Quantification of integrin subunit expression used the antibodies CD11a PE (#101107), CD11b PerCP (#101230), CD11c BV650 (#564079, BD Biosciences), CD18 APC (#562828, BD Biosciences), CD115 BV605 (catalog #135577), and Gr-1 PE/Dazzle 594 (#108452). Briefly, cells were washed with PBS twice, counted, and Fc receptors were blocked with anti-mouse CD16/32 antibody for 10 minutes at 4°C. Cells were washed with PBS, and True-Stain Monocyte Blocker was added before labeling with eBioscience Fixable Viability Dye eFluor 660 (Thermo Scientific) for 30 minutes. Cells were washed with FACS buffer with 0.1% sodium azide (flow buffer), and True-Stain Monocyte Blocker was added again followed by 30-minute incubation with the remaining labeling antibodies. Excess antibodies were washed and cells were fixed with flow cytometry buffer supplemented with 1% paraformaldehyde prior to flow cytometric analyses. Flow cytometry data was read using a BD LSR-Fortessa-HTS analyzer (BD Biosciences) and processed with the FlowJo software (Ashland, OR).
Martinez L., Li X., Ramos-Echazabal G., Faridi H., Zigmond Z.M., Falcon N.S., Hernandez D.R., Shehadeh S.A., Velazquez O.C., Gupta V, & Vazquez-Padron R.I. (2020). A Novel Genetic Model of Constitutively Active Integrin CD11b/CD18. Journal of immunology (Baltimore, Md. : 1950), 205(9), 2545-2553.
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