Fixable live dead stain

Manufactured by Thermo Fisher Scientific

Sourced in United States, United Kingdom

The Fixable Live/Dead Stain is a cell viability dye used to distinguish live and dead cells in flow cytometry and microscopy applications. It provides a simple and effective way to assess cell health and monitor cellular responses to various treatments or environmental conditions.

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Lab products found in correlation

Lsrfortessa, by BD (8 mentions) Fortessa x20, by BD (6 mentions) Facsdiva 8, by BD (5 mentions) Permeabilization buffer, by Thermo Fisher Scientific (5 mentions) Foxp3 transcription factor staining buffer set, by Thermo Fisher Scientific (4 mentions) Cytofix, by BD (3 mentions) Cytofix cytoperm, by BD (3 mentions) Foxp3 intranuclear staining buffer kit, by Thermo Fisher Scientific (3 mentions) Cell stimulation cocktail, by Thermo Fisher Scientific (3 mentions) Fc block, by Thermo Fisher Scientific (3 mentions) Brilliant violet staining buffer, by BD (2 mentions) 96 well plate, by Sarstedt (2 mentions) Fc receptor blocking reagent, by Miltenyi Biotec (2 mentions) Facscanto 2, by BD (2 mentions) Brefeldin a, by Merck Group (2 mentions) Cxcl10, by Thermo Fisher Scientific (2 mentions) Mem non essential amino acid, by Thermo Fisher Scientific (2 mentions) Tgf β1, by R&D Systems (2 mentions) Tgfβrii, by R&D Systems (2 mentions) Ifn γ xmg1.2, by BioLegend (2 mentions)

Close spelling variants of this product

LIVE/DEAD Fixable Violet Dead Cell Stain Kit (172 citations) LIVE/DEAD stain (166 citations) LIVE/DEAD Fixable Violet Dead Cell Stain (85 citations) Live/Dead Fixable Far Red Dead Cell stain (24 citations) LIVE/DEAD Fixable Far Red stain (8 citations) LIVE/DEAD Fixable Violet Dead Stain Kit (7 citations) LIVE/DEAD™ Fixable Dead Cell Stain Sampler Kit (6 citations) Live/Dead Fixable Blue Cell Stain Kit (5 citations) Live–Dead Fixable Dead Cell Stain near-IR-fluorescent reactive dye (4 citations) Aqua LIVE/DEAD® Fixable Aqua Dead Cell Stain Kit (4 citations)

61 protocols using fixable live dead stain

Tumor Immune Profiling in Mice

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Tumor tissues were resected from mice and minced into small pieces and then were lysed by 1 mg/ml collagenase IV (Sigma, United States) and DNase I (Invitrogen, United States) for 1 h at 37°C. Afterward, the tissue medium was filtrated using a 70-μm filter screen to obtain single-cell suspensions. The cell suspensions were stained with antibodies for 30 min and washed three times by PBS and then were subjected to FCM analysis. The following reagents and antibodies were used in FCM analysis.
Panel A: LIVE/DEAD™ Fixable Stain (Invitrogen, California, United States), anti-mouse CD45-BV605 (Biolegend, California, United States), anti-mouse CD3-PE-cy7 (Biolegend, California, United States), anti-mouse CD4-Efluor450 (BD, New Jersey, United States), anti-mouse CD8-Percp-cy5.5 (Biolegend, California, United States), anti-mouse CD19-BV650 (Biolegend, California, United States), and anti-mouse NK1.1-PE (Biolegend, California, United States).
Panel B: LIVE/DEAD™ Fixable Stain (Invitrogen, California, United States), anti-mouse CD45-BV605 (Biolegend, California, United States), anti-mouse CD11b-Percp-cy5.5 (Biolegend, California, United States), anti-mouse F4/80-PE (Biolegend, California, United States), and anti-mouse Ly6G-APC (Biolegend, California, United States).

Liu Z.Y., Zhang D.Y., Lin X.H., Sun J.L., Abuduwaili W., Zhang G.C., Xu R.C., Wang F., Yu X.N., Shi X., Deng B., Dong L., Weng S.Q., Zhu J.M., Shen X.Z, & Liu T.T. (2022). Nalidixic acid potentiates the antitumor activity in sorafenib-resistant hepatocellular carcinoma via the tumor immune microenvironment analysis. Frontiers in Pharmacology, 13, 952482.

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Multiparameter Flow Cytometry Staining

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All samples were treated with Fc receptor blocking reagent (Miltenyi Biotec) before staining. Surface staining was performed in 96-well plates (Sarstedt) in staining buffer of 50% PBS, 50% Brilliant Violet staining buffer (BD). Fixable live/dead stain (Life Technologies) was added to the staining buffer. Antibody staining was conducted for 15 min at 37°C in the dark before washing with PBS. Samples for surface staining only were fixed in Cytofix (BD). Samples for intracellular staining were fixed in Cytofix/cytoperm (BD) for 20 min at 4°C in the dark before staining with intracellular antibodies in saponin buffer [PBS + 1% FBS (Sigma) + 0.1% saponin (Sigma)] for 30 min at 4°C in the dark. Samples were then washed once in saponin buffer and once in PBS. Intranuclear staining was performed using FoxP3 staining buffer (BD). Surface staining was as above, then cells were fixed in buffer A for 10 min at room temperature followed by buffer A with 1:50 buffer B for 30 min at room temperature. Samples were washed in PBS and intranuclear staining performed in PBS for 30 min at 4°C in the dark. Samples were acquired on Fortessa X20 (BD) and data analyzed in FlowJo X (TreeStar).

Easom N.J., Stegmann K.A., Swadling L., Pallett L.J., Burton A.R., Odera D., Schmidt N., Huang W.C., Fusai G., Davidson B, & Maini M.K. (2018). IL-15 Overcomes Hepatocellular Carcinoma-Induced NK Cell Dysfunction. Frontiers in Immunology, 9, 1009.

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Immunophenotyping of Blood Leukocytes

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Blood, collected from buccal sinus and treated with heparin (10 U/mL), was incubated with 1× RBC lysis buffer (Biolegend, San Diego, CA, USA) and then was resuspended in 100 μL of FACS buffer (PBS with 2% FBS), with Fixable Live/Dead Stain (Life Technologies, Carlsbad, CA, USA) and an appropriate combination of fluorescent antibodies specific to CD45, F4/80, CD3, CD45R (Thermo Fischer, Waltham, MA, USA), and FcR block (Biolegend, San Diego, CA, USA). After that, cells were incubated with BTN-Kat and ITN-Kat on ice without permeabilization, or stained by intracellular cytokine staining protocol using BD Fixation and Permeabilization Solution Kit with BD GolgiPlug™ (Becton Dickinson, Franklin Lakes, NJ, USA). Data were analyzed using CytExpert 2.0 (Beckman Coulter, Brea, CA, USA).

Gorshkova E.N., Efimov G.A., Ermakova K.D., Vasilenko E.A., Yuzhakova D.V., Shirmanova M.V., Mokhonov V.V., Tillib S.V., Nedospasov S.A, & Astrakhantseva I.V. (2018). Properties of Fluorescent Far-Red Anti-TNF Nanobodies. Antibodies, 7(4), 43.

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Analysis of Lung Cancer Tumor Samples

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Surgical resection samples from lung cancer patients were analyzed by Caprion Biosciences Inc. (Québec, Canada). This work was approved by the Comité éthique en recherche du Center Hospitalier de l'Université de Montréal and informed consent was obtained using informed consent forms. The tumor samples were fragmented using a scalpel and a single-cell suspension was obtained by tissue disaggregation in a Medimachine system (BD Biosciences), per manufacturer's instructions. Cell suspensions were filtered using a 100-µm pore size, washed in PBS and filtered again using a 40-µm pore size. Cells were then stained with fixable live/dead stain (Life Technologies) and fluorophore conjugated antibodies against CD4⁺, CD8⁺, CD3 (BD Biosciences), FoxP3 and GITR (Biolegend). Stained cells were acquired using a BD LSR Fortessa cytometer and resulting data analyzed using FlowJo.

Tigue N.J., Bamber L., Andrews J., Ireland S., Hair J., Carter E., Sridharan S., Jovanović J., Rees D.G., Springall J.S., Solier E., Li Y.M., Chodorge M., Perez-Martinez D., Higazi D.R., Oberst M., Kennedy M., Black C.M., Yan L., Schwickart M., Maguire S., Cann J.A., de Haan L., Young L.L., Vaughan T., Wilkinson R.W, & Stewart R. (2017). MEDI1873, a potent, stabilized hexameric agonist of human GITR with regulatory T-cell targeting potential. Oncoimmunology, 6(3), e1280645.

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Multiparametric Flow Cytometry Analysis

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All samples were treated with Fc receptor blocking reagent (Miltenyi Biotec) before staining. Surface staining was performed in 96 well plates (Sarstedt) in staining buffer of 50% PBS, 50% Brilliant Violet staining buffer (BD). Fixable live/dead stain (Life Technologies) was added to the staining buffer. Antibody staining was conducted for 15 min at 37°C in the dark before washing with PBS using the following monoclonal antibodies: CD3 PE-Cy7 and CD8 Alexa 700 (eBioscience), CD56 ECD (Beckman Coulter), NKG2D Alexa 488 (Biolegend). Cell viability was determined and dead cells excluded using fixable live/dead aqua stain (Invitrogen). Samples for were fixed in Cytofix (BD). Single fluorochrome compensation controls were made using compensation beads (BD). Compensation matrices were calculated initially in FACSDiva and edited where necessary in FlowJo X (TreeStar). Samples were acquired on LSR Fortessa (BD) and data analyzed in FlowJo X.

Easom N.J., Marks M., Jobe D., Gillmore R., Meyer T., Maini M.K, & Njie R. (2020). ULBP1 Is Elevated in Human Hepatocellular Carcinoma and Predicts Outcome. Frontiers in Oncology, 10, 971.

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Isolation and Characterization of Immune Cells

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Single-cell suspensions of BM were obtained by crushing femurs, tibias, iliac crests, and sternum with mortar and pestle. BM cells were then filtered on a 40-μm cell strainer. A proportion of the BM cells was frozen for later use in in vitro cultures. Spleen, MLN, PP, thymus, and peritoneal cavity single-cell suspensions were obtained by gently pushing cells through a 40-μm cell strainer with a syringe. All cells were stained for extracellular markers and dead cells were identified with fixable live/dead stain (Ebioscience, San Diego, CA, USA), after which intracellular staining was enabled by fixing and permeabilizing cells with Fix/Perm buffer (Ebioscience) according to manufacturer’s instructions. Antibodies used for flow cytometric measurements are listed in Supplementary Table 1 in Data Sheet 1. All flow cytometric measurements were performed on a Canto II flow cytometer (BD Biosciences, Erembodegem, Belgium). FlowJo vX.07 software (Tree Star) was used for data analysis. Gating of all presented immune cell populations was based on single live cells.

van Beek A.A., Sovran B., Hugenholtz F., Meijer B., Hoogerland J.A., Mihailova V., van der Ploeg C., Belzer C., Boekschoten M.V., Hoeijmakers J.H., Vermeij W.P., de Vos P., Wells J.M., Leenen P.J., Nicoletti C., Hendriks R.W, & Savelkoul H.F. (2016). Supplementation with Lactobacillus plantarum WCFS1 Prevents Decline of Mucus Barrier in Colon of Accelerated Aging Ercc1−/Δ7 Mice. Frontiers in Immunology, 7, 408.

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Flow Cytometric Analysis of CD8+ T Cells

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For the detection of CD8+ T cell expansion in the blood, lymphocytes were washed and incubated with 10 mg/mL 2.4G2 FcR block for 15 min at 4 C. Fluorochrome-labeled antibodies to surface markers were then added for 20-30 min in PBS + 1% BSA and the cells washed once with PBS/1% BSA. Red blood cells were lysed with either ammonium chloride buffer (in house) or with Erythrolyse Red Blood cell lysis buffer (AbD SeroTec) and samples washed once with PBS/1% BSA. Samples were then run on a BD FACSCanto II or FACSCalibur and data analyzed using FCS Express or FACs Diva software. Staining of lymphocytes from spleens and tumors were performed similarly. In some cases, tumor samples were additionally stained with a fixable live/dead stain (eBioscience) to visualize only the live cell population. For intracellular staining, cells were surface stained and then fixed and intracellularly stained using the anti-Mouse/Rat Foxp3 Staining Set (BD Biosciences). Antibodies were anti-CD4 eF450 (GK1.5), anti-CD8-APC-eF780 or -FITC (53-6.7), anti-Foxp3 APC or -PE (FJK-16), anti-4-1BB-PE or -eF450 (17-B5), anti-KLRG-1-APC (2F1), anti-MHCII-APC-eF780 (M5/114.15.2), anti-CD11c-APC (N418), streptavidin-PE-Cy7 (all eBioscience), anti-Ki67 APC (B56) (BD Biosciences), anti-4-1BBbiotin (Biolegend) or isotype controls.

Buchan S.L., Dou L., Remer M., Booth S.G., Dunn S.N., Lai C., Semmrich M., Teige I., Mårtensson L., Penfold C.A., Chan H.T.C., Willoughby J.E., Mockridge C.I., Dahal L.N., Cleary K.L.S., James S., Rogel A., Kannisto P., Jernetz M., Williams E.L., Healy E., Verbeek J.S., Johnson P.W.M., Frendéus B., Cragg M.S., Glennie M.J., Gray J.C., Al-Shamkhani A, & Beers S.A. (2018). Antibodies to Costimulatory Receptor 4-1BB Enhance Anti-tumor Immunity via T Regulatory Cell Depletion and Promotion of CD8 T Cell Effector Function. Immunity, 49(5).

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Bacterial Media Preparation and Staining

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Bacterial media MHB and MHB2 were purchased from Millipore Sigma (St. Louis, MO, USA). PI and fixable live/dead stain were obtained from Thermo Fisher Scientific (Waltham, MA, USA). Peptide stock solution was prepared autoclaved PBS (pH 7.4) and filter-sterilized unless stated otherwise.

Di Y.P., Lin Q., Chen C., Montelaro R.C., Doi Y, & Deslouches B. (2020). Enhanced therapeutic index of an antimicrobial peptide in mice by increasing safety and activity against multidrug-resistant bacteria. Science Advances, 6(18), eaay6817.

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Evaluating Stress-Induced GRP78 Expression

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In vitro grown B16F1 and MeWo tumour lines untreated or treated for 5hrs at 37°C with 2µM thapsigargin (Sigma-Aldrich) or SKOV3 and MCF7 tumour lines were stained with citrullinated GRP78 antibody at 15µg/ml or isotype control followed by a FITC conjugated anti-mouse IgG secondary antibody (Sigma-Aldrich) in combination with a fixable live/dead stain (Thermofisher) and using intracellular fixation and permeabilization buffer set (Thermofisher). In vivo grown B16F1 tumours were mechanically disaggregated and stained as above with inclusion of surface stain for CD45 (anti-mouse CD45 efluor450, Thermofisher).

Brentville V.A., Symonds P., Chua J., Skinner A., Daniels I., Cook K.W., Koncarevic S., Martinez-Pinna R., Shah S., Choudhury R.H., Vaghela P., Weston D., Al-Omari A., Davis J, & Durrant L.G. (2022). Citrullinated glucose-regulated protein 78 is a candidate target for melanoma immunotherapy. Frontiers in Immunology, 13, 1066185.

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Multiparametric Flow Cytometry Analysis

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For phenotypic analysis of lymphocyte subsets by multi-color flow cytometry, PBMCs were stained with fluorochrome-conjugated antibodies for the surface markers CD3 (OKT3, Biolegend), CD4 (SK3, Biolegend), CD8 (SK1, Biolegend), CD14 (M5E2, Biolegend), CD19 (HIB19, Biolegend), CD56 (NCAM16.2, BD Biosciences), CD94 (DX22, Biolegend), CD161 (HP-3G10, Biolegend), CRACC (235614, R&D Systems) NKG2A (Z199, Beckman Coulter), NKp80 (MA152, Beckman Coulter), and TCRγδ (IMMU510, Beckman Coulter) as well as for intracellular EOMES (WD1928, eBioscience), granulysin (GNLY) (DH2, Biolegend), NKG7 (2G9, Beckman Coulter), and PLZF (R17-809, BD Biosciences). Intracellular HOPX was detected with an unconjugated primary antibody (rabbit polyclonal, Proteintech) followed by a fluorochrome-labeled F(ab)₂ anti-rabbit IgG secondary antibody (goat polyclonal, ThermoFisher). Dead cells were identified using a fixable live/dead stain (ThermoFisher).

Anderson J., Olafsdottir T.A., Kratochvil S., McKay P.F., Östensson M., Persson J., Shattock R.J, & Harandi A.M. (2018). Molecular Signatures of a TLR4 Agonist-Adjuvanted HIV-1 Vaccine Candidate in Humans. Frontiers in Immunology, 9, 301.

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