Zombie uv dye

Manufactured by BioLegend

Sourced in United States

Zombie UV dye is a fluorescent dye that binds to nucleic acids in dead cells, emitting a bright fluorescent signal when exposed to UV light. It can be used to identify and quantify dead cells in a sample.

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Close spelling variants of this product

Zombie UV (60 citations) Zombie dye (26 citations) Zombie UV Fixable Viability Dye (21 citations) Zombie UV viability dye (16 citations) Zombie UV Live/Dead dye (14 citations) Zombie UV fixable viability dye kit (6 citations) Live/Dead stain (Zombie UV Dye: (3 citations) Primary antibodies for SERINC5 and cell viability dye Zombie UV (2 citations) Zombie-UV staining Dye (2 citations) Cell viability dye Zombie UV (2 citations)

29 protocols using zombie uv dye

Isolation and Characterization of Mouse Skin Immune Cells

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For the analysis of mouse skin, 1 cm × 1 cm dorsal skin was cut off and transferred to an EP tube containing 1 mL Hank’s Balanced Saline Solution (HBSS, H4641, Sigma-Aldrich). The skin was washed rigorously by quickly shaking up and down by hand for 15 s × 3 times. The skin was cut into pieces (<0.5 mm in size) in a 6-well plate placed on ice with dulbecco’s modified eagle medium (DMEM, 11885-084, Gibco) (not supplemented with FBS) containing 1 mg/mL Collagenase P (11213857001, Roche, USA) and 0.2 mg/mL DNase I (AMPD1, Sigma-Aldrich). The samples were incubated in a 37 °C cell culture incubator for 60 min and pipetted every 20 min to gently mix the cells. Cell suspensions were filtered through 40 μm cell strainer. Cells were stained with PerCP/Cy5.5 anti-mouse CD45 (103132, 1:100, BioLegend), FITC anti-mouse Ly6G (127606, 1:100, BioLegend), PE anti-mouse CXCR4 (146506, 1:100, BioLegend), and Zombie UV dye (423102, 1:500, BioLegend) in FACS buffer for 30 min and then analyzed by flow cytometry (649225, BD LSRFortessa Cell Analyzer). Data were analyzed with Flowjo v10.8.1 (Tree Star). Background fluorescence levels were determined by Fluorescence Minus One (FMO).

Chen J., Bai Y., Xue K., Li Z., Zhu Z., Li Q., Yu C., Li B., Shen S., Qiao P., Li C., Luo Y., Qiao H., Dang E., Yin W., Gudjonsson J.E., Wang G, & Shao S. (2023). CREB1-driven CXCR4hi neutrophils promote skin inflammation in mouse models and human patients. Nature Communications, 14, 5894.

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Cytokine Expression Profiling of Hepatic Immune Cells

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Single cell suspensions were derived from hepatic homogenate and stained with directly conjugated monoclonal antibodies or isotype controls. Staining for cytokine expression was completed after 4 hours of stimulation with 50 ng/mL Phorbol 12-Myristate 13-acetate (PMA; Promega), 1 μg/mL Ionomycin (Calbiochem) and 10μg/mL Brefeldin A (Gold Bio). Data collection and analysis were done as previously described^{11 (link),63 (link)}. Briefly, cells were stained with Live/Dead stain (Zombie UV Dye: Biolegend) and with directly-conjugated monoclonal antibodies to CD45-AF700 (104), CD11b-PE (M1/70), F4/80-APCef780 (BM8), Ly6C-Percp (HK1.4), Gr1-FITC (RB6-8C5), NK1.1-PECy7 (PK136), TCRβ-PE (H57-597), CD4-APCef780 (GK1.5), CD8-ef450 (53-6.7), TNF-BV650 (MP6-XT22), IL-17A-Percp (17B7) and IL-17F-PE (18F10) [all antibodies from e-Bioscience]. Flow cytometry data were then collected using a LSR Fortessa (BD) flow cytometer and analyzed using FlowJo X software (vX0.7).

Giles D.A., Moreno-Fernandez M.E., Stankiewicz T.E., Graspeuntner S., Cappelletti M., Wu D., Mukherjee R., Chan C.C., Lawson M.J., Klarquist J., Sünderhauf A., Softic S., Kahn C.R., Stemmer K., Iwakura Y., Aronow B.J., Karns R., Steinbrecher K.A., Karp C.L., Sheridan R., Shanmukhappa S.K., Reynaud D., Haslam D.B., Sina C., Rupp J., Hogan S.P, & Divanovic S. (2017). Thermoneutral housing exacerbates non-alcoholic fatty liver disease in mice and allows for sex-independent disease modeling. Nature medicine, 23(7), 829-838.

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Evaluating CX-6258 Cytotoxicity on Tumor-Infiltrating Lymphocytes

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Human Tumor infiltrating lymphocytes (TILs) from melanoma patients were cultured and expanded as previously described(20 (link)). To assess cytotoxicity of CX-6258 on human TILs, cells were seeded in RPMI with 10% human AB serum, 3000 IU/ml hIL2 at 1×10⁵ cells per well of a 96 round bottom plate (Corning) and treated with increasing doses of CX-6258 in triplicates. After 48 hours cells were collected, washed once with PBS and resuspended in Annexin V staining buffer. Cells were stained with Annexin V-FITC and Propidium iodide (PI) (Cell Signaling) and analyzed on a Sony SP6800 Spectral Flow Cytometer. Data were analyzed using FlowJo 10.5.3 (TreeStar) and viable cells defined by negative staining for Annexin V and PI are reported. For proliferation assays 5×10⁴ TILs were stained with CFSE (Invitrogen) and seeded together with anti-CD3/CD28 Beads (Invitrogen) and increasing doses of CX-6258 in T-cell media supplemented with 30 IU/ml IL2. Cells were collected on days 1,3 and 5, stained for viability using Zombie UV dye (BioLegend) and fixed using 4 % formaldehyde in PBS. Cells were analysed by FACS on a BD-Fortessa (Becton Dickinson, Franklin Lakes, NJ, USA). Data analysis was performed with FlowJo 10.5.3 (TreeStar) and viable cells were defined by negative staining for Zombie and analyzed for CFSE intensity.

Melms J.C., Vallabhaneni S., Mills C.E., Yapp C., Chen J.Y., Morelli E., Waszyk P., Kumar S., Deming D., Moret N., Rodriguez S., Subramanian K., Rogava M., Cartwright A.N., Luoma A., Mei S., Brinker T.J., Miller D.M., Spektor A., Schadendorf D., Riggi N., Wucherpfennig K.W., Sorger P.K, & Izar B. (2019). Inhibition of Haspin kinase promotes cell-intrinsic and extrinsic anti-tumor activity. Cancer research, 80(4), 798-810.

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Comprehensive Immunophenotyping of Cryopreserved TILs

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Cryopreserved TILs were thawed in warmed RPMI plus benzonase (MilliporeSigma) and washed in RPMI. Cells were plated in a round-bottom 96-well plate at 1–2e6 cells/well. Cells were washed with PBS, blocked with mouse CD16/32 Fc block (BioLegend), and then stained with Zombie UV dye (BioLegend) and surface antibody cocktail, then permeabilized with eBioScience FoxP3 Fixation/Permeabilization kit (eBioscience, Waltham, MA, USA) according to manufacturer’s protocol. They were stained with intracellular antibody cocktail, then fixed with 2% paraformalin and kept on ice until acquisition on a BD Symphony A3 (BD). Data were compensated for fluorescent spillover and analyzed using FlowJo 10.8.1 (RRID: SCR_008520) (BD).

Watters C.R., Barro O., Elliott N.M., Zhou Y., Gabere M., Raupach E., Baker A.T., Barrett M.T., Buetow K.H., Jacobs B., Seetharam M., Borad M.J, & Nagalo B.M. (2023). Multi-modal efficacy of a chimeric vesiculovirus expressing the Morreton glycoprotein in sarcoma. Molecular Therapy Oncolytics, 29, 4-14.

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Multiparametric Flow Cytometry of Immune Cells

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Flow cytometry was performed from organ single-cell suspensions or purified neutrophils, live cells were identified using zombie UV dye (#423108 BioLegend) or live/dead blue (#L34961 Invitrogen), intracellular cytokine staining was performed following 4 h restimulation with phorbol myristate acetate (PMA; 50 ng/mL) and ionomycin (500 ng/mL), followed by a 1h Brefeldin A (5 µg/ml) incubation. Antibodies: (all from BioLegend unless stated otherwise): CD11b FITC (1:200, M1/70), CD11b PerCP (1:100 M1/70), CD11b Bv510 (1:100, M1/70), Ly6C PerCP/Cy5.5 (1:400, HK 1-4), Ly6C PE (1:800, HK 1-4), Ly6G APC/Cy7 (1:400, 1A8), Ly6G PE (1:400, 1A8), Ly6G Bv785 (1:100, 1 A8), PD-L1 PE (1:100, 10F.9G2), CD54 AF647 (1:200, YN1/1.74), CD117 BUV395 (1:100, 2B8, BD), TNF-α BV421 (1:200, MP6-XT22), CCL2 PE (1:50, 2H5), CXCL1 AF647 (1:50, 1174A, R&D). Human neutrophils were analyzed in peripheral blood or after isolation using the MACSxpress^® kit (Miltenyi Biotech). Isolated cells were stimulated with PMA (10 ng/mL), LPS (0.1 µl/mL) and CL097 (1µg/mL) for 4 hours. Antibodies: CD16-APC-Cy7 (1:200, 3G8) and CD66B-APC (1:400, G10F5, Biolegend), viperin (RSAD2) PE (1:200, MaP,VIP; Biosience). Flow cytometry analysis was performed on a Cytek Aurora (Cytek)- or BD LSRII cytometer and data analysis were performed using FlowJo V10.4.2 software.

Er-Lukowiak M., Hänzelmann S., Rothe M., Moamenpour D.T., Hausmann F., Khatri R., Hansen C., Boldt J., Bärreiter V.A., Honecker B., Bea A., Groneberg M., Fehling H., Marggraff C., Cadar D., Bonn S., Sellau J, & Lotter H. (2023). Testosterone affects type I/type II interferon response of neutrophils during hepatic amebiasis. Frontiers in Immunology, 14, 1279245.

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Intradermal Ear Inflammation Assay

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Ansamitocin-P3 (4 μg/ear) or LPS (8 μg/ear) or Vehicle (1.5% DMSO) was injected intradermally into the ears of C57BL/6N WT or GEFH1^−/− mice. Analysis was performed after 24 hours using flow cytometry. Epidermal sheets were digested with Accutase (Sigma), collagenase IV (Worthington), hyaluronidase (Sigma), and DNase type IV (Sigma). Single-cell suspensions were prepared and stained with anti-CD45, anti-CD11c, anti-MHC-II, anti-CD86 and anti-CD80 antibodies. Dead cells were excluded using Zombie UV dye (BioLegend).

Kashyap A.S., Fernandez-Rodriguez L., Zhao Y., Monaco G., Trefny M.P., Yoshida N., Martin K., Sharma A., Olieric N., Shah P., Stanczak M., Kirchhammer N., Park S.M., Wieckowski S., Laubli H., Zagani R., Kasenda B., Steinmetz M.O., Reinecker H.C, & Zippelius A. (2019). GEF-H1 Signaling upon Microtubule Destabilization Is Required for Dendritic Cell Activation and Specific Anti-tumor Responses. Cell reports, 28(13), 3367-3380.e8.

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Cytokine Profiling of Immune Cells

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Mouse cells were stained with Live/Dead stain (Zombie UV Dye: Biolegend) and with directly conjugated monoclonal antibodies to CD45-PE-Dazzle594 (Biolegend, 104), NK1.1-BV711 (Biolegend, PK136) then fixed, permeabilized and stained for the cytokine IFNγ-PE-Cy7 (e-Biosciences, XMG1.2).

Oates J.R., Sawada K., Giles D.A., Alarcon P.C., Damen M.S., Szabo S., Stankiewicz T.E., Moreno-Fernandez M.E, & Divanovic S. (2023). Thermoneutral housing shapes hepatic inflammation and damage in mouse models of non-alcoholic fatty liver disease. Frontiers in Immunology, 14, 1095132.

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Multi-parameter Immune Cell Analysis

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Cells were stained for viability with Zombie-NIR dye. Cell surface staining was performed in cell staining media (PBS with 0.5% BSA and 0.02% NaN3) for 30 minutes at room temperature. The following anti-mouse antibodies were used: TCRβ – APC (H57–597), CD8+ – PE (53–5.8), CD62L - BV421 (MEL-14), KLRG1 – BV510 (2F1/KLRG1), CD44 – PE-Cy7 (IM7), CD25 – FITC (3C7), CD19 – APC-Cy7 (1D3/CD19), F480 APC-Cy7 (BM8). Stained cells were analyzed with an LSR II flow cytometer (BD Biosciences). MitoTracker Deep Red (Thermo Fisher, Waltham, Massachusetts) staining was performed per manufacturer’s instructions and as previously (Scharping et al, 2016). For MitoTracker Deep Red experiments, Zombie-UV dye was used (Biolegend, San Diego, California).
For sorting experiments, cells were prepared as described for flow cytometry and then sorted into FBS containing media (RPMI 1640, 20% FBS, 1% HEPES, 100 mg/mL penicillin/streptomycin) on a FACSAria II (BD Biosciences).

Levine L.S., Hiam-Galvez K.J., Marquez D.M., Tenvooren I., Madden M.Z., Contreras D.C., Dahunsi D.O., Irish J.M., Oluwole O.O., Rathmell J.C, & Spitzer M.H. (2021). Single-cell analysis by mass cytometry reveals metabolic states of early activated CD8+ T cells during the primary immune response. Immunity, 54(4), 829-844.e5.

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Single-cell analysis of skin immune cells

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For in vitro human studies, cells were harvested from cell culture, stained with primary antibodies on ice for 30 min, washed, stained with 7-AAD (BioLegend), and acquired on a BD Fortessa X-20. For animal studies, ear skin was digested in 500 μl of Liberase TL (0.25 mg/ml) (Roche) in Dulbecco’s modified Eagle’s medium (Sigma-Aldrich) at 37°C and 5% CO₂ for 90 min. Skin and draining lymph nodes were then manually homogenized through a 70-μm cell strainer to obtain a single-cell suspension. All cells were stained with Zombie UV dye (BioLegend) for viability at room temperature for 20 min, followed by primary antibodies on ice for 30 min (table S13). Secondary streptavidin-conjugated fluorophores were stained on ice for 30 min. Cells were then fixed with BD Cytoperm/Cytofix reagent on ice for 30 min or overnight at 4°C before data acquisition on a BD LSRFortessa X-20 special order research product. Data were analyzed with FlowJo 10 (Tree Star).

Mack M.R., Brestoff J.R., Berrien-Elliott M.M., Trier A.M., Yang T.L., McCullen M., Collins P.L., Niu H., Bodet N.D., Wagner J.A., Park E., Xu A.Z., Wang F., Chibnall R., Council M.L., Heffington C., Kreisel F., Margolis D.J., Sheinbein D., Lovato P., Vivier E., Cella M., Colonna M., Yokoyama W.M., Oltz E.M., Fehniger T.A, & Kim B.S. (2020). Blood natural killer cell deficiency reveals an immunotherapy strategy for atopic dermatitis. Science translational medicine, 12(532), eaay1005.

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Multiparameter Flow Cytometry of Immune Cells

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Mouse cells were stained with Live/Dead stain (Zombie UV Dye: Biolegend) and with directly-conjugated monoclonal antibodies to CD45-PE-Dazzle594 (Biolegend, 104), TCRβ-APCef780 or APC (Invitrogen, H57-597), CD8-BV510 (Biolegend, 53-6.7), then fixed, permeabilized and stained for the cytokines IL-17A-PerCpCy5.5 or PE (e-Biosciences, 17B7), IFNγ-PE-Cy7 (e-Biosciences, XMG1.2) and TNFα-BV650 (Biolegend, MP6-XT22).

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