Zombie yellow fixable viability dye

Manufactured by BioLegend

Zombie Yellow fixable viability dye is a fluorescent dye used for the detection of dead or dying cells in flow cytometry and microscopy applications. It is a cell-impermeant dye that can only enter cells with compromised membranes, allowing for the identification of non-viable cells.

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Zombie Yellow (44 citations) Fixable viability dye (27 citations) Zombie dye (26 citations) Zombie viability dye (23 citations) Viability dye (15 citations) Zombie yellow dye (11 citations) Zombie Yellow viability dye (7 citations)

5 protocols using zombie yellow fixable viability dye

Multicolor Flow Cytometry Immunophenotyping

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Approximately 5x10⁵ cells were washed in PBS, resuspended in 100 μl PBS containing 0.1 μl Zombie Yellow fixable viability dye (Biolegend) and incubated for 15 mins at 4°C. Cells were then washed, resuspended in 20 μl of staining buffer containing surface antibodies at previously determined optimum dilution along with 1 μl of human Fc receptor blocking solution for 20 mins at 4°C, fixed in 50 μl of 1% paraformaldehyde solution for 15 mins at room temperature, washed, resuspended in 15 μl of BD Perm/Wash buffer 1x (BD Biosciences) containing intracellular antibodies at previously determined optimum dilution for 30 mins at 4°C, washed with BD Perm/Wash 1x solution, and resuspended in staining buffer. Samples were acquired using a LSR Fortessa 4 (BD Biosciences) with Diva software, and analyzed using FlowJo software (version 6.0).

Burel J.G., Apte S.H., Groves P.L., Klein K., McCarthy J.S, & Doolan D.L. (2016). Reduced Plasmodium Parasite Burden Associates with CD38+ CD4+ T Cells Displaying Cytolytic Potential and Impaired IFN-γ Production. PLoS Pathogens, 12(9), e1005839.

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Neutrophil Identification by Flow Cytometry

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Cells were incubated with CD11b-PE (Clone:M1/70) and Ly6G-APC (Clone: RB6-8C5) for 45 min, all from liankebio (Hangzhou, China). Live versus dead cells were stained using Zombie Yellow™ Fixable viability dye (BioLegend, CA). The data were acquired with a BD FACSCelesta flow cytometer (San Jose, CA) and analyzed by FlowJo software version 10.7.2 (Tree Star Inc., Ashland, OR). Neutrophils were gated as CD11b+Ly6G+ live cells.

Wang Z., Zhao X., Zhou H., Che D., Du X., Ye D., Zeng W, & Geng S. (2023). Activation of ryanodine-sensitive calcium store drives pseudo-allergic dermatitis via Mas-related G protein-coupled receptor X2 in mast cells. Frontiers in Immunology, 14, 1207249.

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Quantifying Neutrophil Recruitment in Peritonitis

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To quantify the leukocyte recruitment, we performed flow cytometry as described previously (15 (link)). Following induction of peritonitis by SP injection, peritoneal lavage was collected and cells were washed with FACS buffer. Peritoneal cells were treated with CD16/32 Fc block (clone93, cat#101320) for 15 min followed by anti-CD45-FITC (clone 30-F11; cat #103108), CD11b-PerCP-Cy5.5 (clone M1/70, cat#101227), and Ly6G-BV421 (clone 1AB, cat#127628) for 45 min to stain neutrophils. Live versus Dead cells were stained using Zombie Yellow™ Fixable viability dye (Biolegend, CA). The data were acquired with BD LSR II flow cytometer (San Jose, CA) and analyzed by FlowJo software version 10.7.2 (Tree Star Inc., Ashland, OR). Neutrophils were gated as CD45⁺CD11b⁺Ly6G⁺ live cells.

Chaki S., Alkanfari I., Roy S., Amponnawarat A., Hui Y., Oskeritzian C.A, & Ali H. (2022). Inhibition of Orai Channel Function Regulates Mas-Related G Protein-Coupled Receptor-Mediated Responses in Mast Cells. Frontiers in Immunology, 12, 803335.

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Multicolor FACS Analysis of B Cell Subsets

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Cells were resuspended at a concentration of 10⁶ cells/100 μl fluorescence-activated cell sorting (FACS) buffer (1X PBS, 2 mM EDTA, 1% BSA). Cells were stained with Fc block (BD Biosciences) for fifteen minutes, stained with antibody-fluorophores for one hour on ice, then washed with ten volumes of FACS buffer. The following antibodies were used: B220-PE-Cy7 (RA3–6B2, Tonbo), CD11b-APC-Cy7 (M1/70, Tonbo), CD45.1-FITC (A20, Tonbo), CD45.2-PerCP-Cy5.5 (104, Tonbo), CD90.2-APC-Cy7 (30-H12, BioLegend), F4/80-APC-Cy7 (BM8, BioLegend), CD138-BV711 (281–2, BD), CD21-APC (B-ly4, BD), CD43-PE (S7, BD), GL7-eFluor660 (GL-7, eBioscience), CD23-eFluor450 (B3B4, eBioscience), IgM-FITC (II/41, eBioscience), Annexin V-APC (kit number 88–8007-72, eBioscience), and Zombie Yellow Fixable Viability Dye (kit number 423104, BioLegend). An LSRII was used for analysis and an ARIAII was used for sorting (BD Biosciences). All flow cytometry data was analyzed with FlowJo version 9.9.5. Preceding all flow cytometry analyses presented is the following gating strategy: 1) lymphocytes (forward scatter [FSC]-area by side scatter [SSC]-area), 2) singlets (FSC-width by FSC-height), 3) singlets (SSC-width by SSC-height), 4) live cells (viability dye⁻), 5) exclusion of non-B cell lineage cells (Thy1.1⁻F4/80⁻CD11b⁻).

Haines R.R., Barwick B.G., Scharer C.D., Majumder P., Randall T.D, & Boss J.M. (2018). The histone demethylase LSD1 regulates B cell proliferation and plasmablast differentiation. Journal of immunology (Baltimore, Md. : 1950), 201(9), 2799-2811.

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Flow Cytometry Cell Preparation

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On day 6 cells were centrifuged at 500 × g for 5 min to remove supernatant, resuspended in D-PBS, pH 7.4, then transferred to a 96 well plate in preparation for staining with Zombie Yellow fixable viability dye (BioLegend, San Diego, CA) and surface labeling with antibodies. Following labeling with primary, secondary, and directly conjugated antibodies in Table 1, cells were resuspended in 200 μl stabilizing fixative and analyzed using a BD LSR II flow cytometer (BD Biosciences, San Jose, CA). Data were analyzed using FlowJo software (Tree Star, Inc., San Carlos, CA) and cell populations were expressed as a percentage of the live cell population.

Wherry T.L., Mooyottu S, & Stabel J.R. (2022). Effects of 1,25-Dihydroxyvitamin D3 and 25-Hydroxyvitamin D3 on PBMCs From Dairy Cattle Naturally Infected With Mycobacterium avium subsp. paratuberculosis. Frontiers in Veterinary Science, 9, 830144.

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