Live dead yellow

Manufactured by Thermo Fisher Scientific

Live/Dead Yellow is a fluorescent dye used in cell viability assays. It selectively stains dead cells, allowing for the differentiation between live and dead cells in a sample.

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Lab products found in correlation

Lsrfortessa, by BD (6 mentions) Cytofix cytoperm, by BD (5 mentions) Anti cd16 cd32 fc block, by BD (4 mentions) Golgistop, by BD (3 mentions) Fortessa, by BD (3 mentions) Cytofix, by BD (3 mentions) Lsrii flow cytometer, by BD (3 mentions) Anti cd8 pe texas red, by Thermo Fisher Scientific (3 mentions) Anti cd3 percp cy5, by BD (3 mentions) Carboxyfluorescein succinimidyl ester (cfse), by Thermo Fisher Scientific (2 mentions) Golgiplug, by BD (2 mentions) Cd4 percp cy5, by BD (2 mentions) Ifnγ bv650, by BioLegend (2 mentions) Cd107a fitc, by BD (2 mentions) Tnf α pe cy7, by BD (2 mentions) Il 2 pe, by BioLegend (2 mentions) Cd3 apc, by BD (2 mentions) Cd8 apc cy7, by BD (2 mentions) Anti cd4 pacific blue, by Thermo Fisher Scientific (2 mentions) Anti cd28 and anti cd49d, by BD (2 mentions)

Close spelling variants of this product

LIVE/DEAD Fixable Yellow Dead Cell Stain Kit (99 citations) LIVE/DEAD Fixable Yellow Dead Cell Stain (51 citations) LIVE/DEAD Fixable Yellow stain (11 citations) Live/Dead Fixable Yellow dye (10 citations) Live/Dead Yellow stain (5 citations) Live/Dead yellow viability dye (4 citations) Live/Dead fixable yellow dead cell staining kit (3 citations) LIVE-DEAD® Fixable yellow or near-IR dead cell stain kit (2 citations) Aqua or Yellow LIVE/DEAD viability stain (2 citations) Live/Dead Fixable Aqua or Yellow Dead Cell Stain (2 citations)

37 protocols using live dead yellow

T-cell activation with LDNs/NDNs

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1x10⁵ T cells were stained with CFSE (Thermo) and cultured in 200μl RPMI (Gibco) + 10% HI-FCS (Gibco) + 1% Peniciliin/Streptomycin (100x, Gibco) + 1% L –glutamine (100x, Gibco) with 2x104 Human T-Activator CD3/CD28 Dynabeads (Thermo) alone or with 2x105 LDNs or NDNs isolated form the same donor for 96 hours at 37°C 5% CO2. Activation cocktail containing protein transport inhibitors (500x eBioscience) was added for 4 hours then stained with Live/Dead Yellow (Thermo), followed by antibodies for CD4, CD8, CD66b (Biolegend) and intracellular IFNγ (Biolegend) following fixation and permeabilisation with 1x Fix/Perm solution (BD). Cells were then run on an LSR Fortessa flow cytometer (BD) and analysed with FlowJo Software (BD). Co-culture conditions were adapted from previously described methods (34 (link)).

Hardisty G.R., Llanwarne F., Minns D., Gillan J.L., Davidson D.J., Gwyer Findlay E, & Gray R.D. (2021). High Purity Isolation of Low Density Neutrophils Casts Doubt on Their Exceptionality in Health and Disease. Frontiers in Immunology, 12, 625922.

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Enrichment and Annexin V Staining of Murine iNKT Cells

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Thymocyte suspensions were prepared from individual thymi of C57BL/6J mice 8 weeks of age, and enriched for iNKT cells as described for iNKT subset isolation for RNA-Seq analysis. Enriched thymus suspensions were resuspended at 5 × 10⁶ cells/ml in RPMI containing 10% FBS, 50 µM 2-mercaptoethanol and pen-streptomycin-glutamine (Gemini #400–110), and then cultured in 6 well T.C. plates for 4 h at 37 °C before harvesting and staining. Cells were stained for surface proteins, then washed and stained with AnnexinV-FITC (BD Biosciences #556420) or AnnexinV-APC (BD Biosciences #550475) together with Live-Dead Yellow (ThermoFisher Scientific #L34959) in Annexin binding buffer as described in protocols supplied by BD Biosciences.

Engel I., Seumois G., Chavez L., Samaniego-Castruita D., White B., Chawla A., Mock D., Vijayanand P, & Kronenberg M. (2016). Innate-like functions of natural killer T cell subsets result from highly divergent gene programs. Nature immunology, 17(6), 728-739.

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Flow Cytometry Analysis of Immune Cells

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Cryopreserved BAL were thawed, seeded 1.2 million cells per well, and rested overnight at 37C and 5% CO₂. Viable cells were stained using a LIVE/DEAD® Yellow (ThermoFisher®) amine dye then surface stained with CD3-PECF594 (Sp34-2), CD4 eluor605NC (OKT4), CD8-V500 (RPA-T8), CD20-PECy7 (2H7), HLA-DR-BV711 (G46-6), CD14-BV785 (M5E2), CD11b-APC-Cy7 (M1/70). Cells were permeablizied with Cytofix/Cytoperm (BD Biosciences) and stained with an intracellular with Ki67 AF700 (B56) in Perm/Wash^TM Buffer (BD Bioscinces). See S2 Table for antibody concentrations. Cells were then washed with Perm/Wash^TM Buffer (BD Bioscinces) then fixed with 1% paraformaldehyde (Sigma) and collected on an LSR II (BD Bioscinces). Flow cytometry was analyzed in FlowJo (Version 9.7.6, Treestar Inc., Ashland, Oregon). See S2 Fig for gating scheme.

Koday M.T., Leonard J.A., Munson P., Forero A., Koday M., Bratt D.L., Fuller J.T., Murnane R., Qin S., Reinhart T.A., Duus K., Messaoudi I., Hartman A.L., Stefano-Cole K., Morrison J., Katze M.G, & Fuller D.H. (2017). Multigenic DNA vaccine induces protective cross-reactive T cell responses against heterologous influenza virus in nonhuman primates. PLoS ONE, 12(12), e0189780.

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Phenotyping Antigen-Specific B cells

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Freshly isolated peripheral blood mononuclear cells were stained first for viability with Live/dead Yellow (ThermoFisher) and then for markers with the following monoclonal antibodies: IgA (IS11–8E10, Miltenyi), IgD (IA6–2, BD), IgG (G18–145, BD), IgM (MHM-88, Biolegend), CD3 (SK7, BD), CD4 (RPA-T4, BD), CD8 (SK1, BD), CD14 (61D3, eBioscience), CD16 (CB16, eBioscience), CD19 (SJ25C1, BD), CD20 (2H7, BD), CD27 (O323, BioLegend or M-T271, BD), CD38 (HB7, BD), and CD71 (CY1G4, BioLegend. Antigen-specific B cells were detected by staining with RBD conjugated to Alexa Fluor 488 (Protein Labeling Kit, ThermoFisher). RBD was conjugated according to manufacturer’s instructions, with the following changes: protein was labeled at a concentration of 1mg/mL, and incubated for 30 minutes without the addition of bicarbonate. After staining, PBMCs were washed and then fixed for 30 minutes using 2% paraformaldehyde (ThermoFisher). Data were acquired on a BD FACSymphony A5 and analyzed using FlowJo 10.7.1 (BD).

Mantus G., Nyhoff L.E., Kauffman R.C., Edara V.V., Lai L., Floyd K., Shi P.Y., Menachery V.D., Edupuganti S., Scherer E.M., Kay A., McNair N., Anderson E.J., Rouphael N., Ahmed R., Suthar M.S, & Wrammert J. (2021). Evaluation of Cellular and Serological Responses to Acute SARS-CoV-2 Infection Demonstrate the Functional Importance of the Receptor Binding Domain. Journal of immunology (Baltimore, Md. : 1950), 206(11), 2605-2613.

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Flow Cytometry Analysis of Immune Cell Markers

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All staining was performed in FACS buffer (0.5% (m/v) BSA, 2 mM EDTA in PBS). Samples were acquired on a BD LSRFortessa (BD Biosciences) or Cytek Aurora (Cytek). Analysis was performed using FlowJo (FlowJo). To analyze cell surface marker expression, cells were collected, washed with PBS and stained with the viability dye Live/Dead Yellow (Thermo Fisher Scientific) according to the manufacturer’s protocol. Cells were then washed with FACS buffer and stained for surface markers: CD45 (APC-Vio 770, clone REA747), CD3 (PerCP Cy5.5, clone OKT3), CD4 (FITC, clone REA623), CD8 (VioBlue, clone REA734), CD107a (PE-Cy5, clone eBioH4A3), CD279 (PE, clone REA1165) and CD274 (BV711, clone 29E.2A3) for 20 min at 4°C.
To analyze intracellular cytokines, cells were collected, washed with PBS, and stained with the viability dye Live/Dead Yellow. Cells were then washed with FACS buffer and stained for surface markers. Cells were further fixed and permeabilized using Inside stain kit (Miltenyi Biotec), according to the manufacturer’s protocol, and stained for the intracellular cytokines IFN-γ (PE-Vio 770, clone REA600) and tumour necrosis factorα (APC, clone REA656).

Acúrcio R.C., Pozzi S., Carreira B., Pojo M., Gómez-Cebrián N., Casimiro S., Fernandes A., Barateiro A., Farricha V., Brito J., Leandro A.P., Salvador J.A., Graça L., Puchades-Carrasco L., Costa L., Satchi-Fainaro R., Guedes R.C, & Florindo H.F. (2022). Therapeutic targeting of PD-1/PD-L1 blockade by novel small-molecule inhibitors recruits cytotoxic T cells into solid tumor microenvironment. Journal for Immunotherapy of Cancer, 10(7), e004695.

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Comprehensive Multiparametric Immunophenotyping of PBMCs

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As part of the original A5314 study, cryopreserved PBMC specimens from A5314 (n=118) were thawed and stained with LiveDead Yellow (ThermoFisher), CD3-BV711 (clone UCHT1, BD), CD4-AlexaFluor(AF)-700 (RPA-T4, BD), CD8-BrilliantViolet(BV)-785 (RPA-T8, BD), HLA-DR-FITC (L243, BD), CD38-APC (HIT2, BD), CD14-Pacific Blue (M5E2, BD), CD16-APC-Cy7 (3G8, BioLegend), CX3CR1-PerCP-Cy5.5 (2A9-1, BioLegend), and CD69-PE-Cy7 (L78, BD). Additional aliquots of cryopreserved PBMCs from each donor at baseline and week 24 were then thawed and stained with a second panel, consisting of LiveDead Aqua (ThermoFisher), CD3-BV605 (SK7, BD), CD4-BrilliantUltraViolet(BUV)395 (SK3, BD), CD8-APC-Cy7 (SK1, BioLegend), CD45RA-APC (HI100, BD), CD27-BV786 (O323, BioLegend), CD95-PE (DX2, BD), CD127-PECF594 (A0195D5, BioLegend), CD39-PerCPCy5.5 (eBioA1, eBioscience), CD73-BV421 (AD2, BioLegend), Ki67-PECy7 (B56, BD), and Bcl-2-FITC (Bcl-2/100, BD). All researchers remained blinded to the treatment status of the donors during flow cytometry acquisition and gating.

Freeman M.L., Clagett B.M., Moisi D., Yeh E., Morris C.D., Ryu A., Rodriguez B., Stein J.H., Deeks S.G., Currier J.S., Hsue P.Y., Anthony D.D., Calabrese L.H., Ribaudo H.J, & Lederman M.M. (2022). Methotrexate Inhibits T Cell Proliferation but Not Inflammatory Cytokine Expression to Modulate Immunity in People Living With HIV. Frontiers in Immunology, 13, 924718.

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Multiparametric Flow Cytometry Staining

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For staining of cell surface molecules, cells were suspended in staining buffer (PBS, 1% bovine serum albumin (BSA), and 0.01% NaN 3 ) and first stained with using PE-or APCconjugated MR1 tetramers at a dilution of 1:300 in staining buffer for 45 minutes at room temperature followed by surface staining with fluorochrome-conjugated antibody at 0.1-1 μg/10 7 cells. Cells were stained with Live/Dead Yellow (ThermoFisher) at 1:500 and Fc receptors were blocked with 2.4G2 antibody at 1:500 and Free Streptavidin at 1:1000 for 15 min at 4˚C. After washing, cells were stained with cell surface-specific antibodies for 30 minutes on ice. For cytokine staining, cells were previously stimulated with 100 ng/ml of PMA and 1 μg/ml of Ionomycin for 1h at 37˚C and then incubated in GolgiStop and GolgiPlug (both from BD PharMingen) for 2 h at 37˚C. For intracellular staining, cells were fixed with CytoFix (BD) for 20 min, and permeabilized with Perm 1X solution (ThermoFisher) with intracellular antibodies overnight. For high-parameter flow cytometry experiments, data were acquired on Fortessa or Symphony S6 (BD Biosciences), data were processed with DIVA (BD Bioscience) and analyzed with FlowJo v10.7 (BD). Opt-tSNE and UMAP dimensional reduction as well as FlowSOM algorithm clustering of flow cytometry data was performed in OMIQ software (OMIQ Inc.).

Chandra S., Ascui G., Riffelmacher T., Chawla A., Ramírez-Suástegui C., Castelan V.C., Seumois G., Simon H., Murray M., Seo G., Premlal A.L.R., Verstichel G., Li Y., Lin C., Greenbaum J., Lamberti J.J., Murthy R., Nigro J.J., Cheroutre H., Ottensmeier C.H., Hedrick S.Μ., Lu L., Vijayanand P., & Kronenberg M. (2021). Transcriptomes and metabolism define mouse and human MAIT cell heterogeneity.

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Phenotypic Characterization of hiPSC-Fibroblasts

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Phenotypic analysis of hiPSC-fibs was performed using multicolor flow cytometry. Briefly, nonspecific binding of antibodies by Fc receptors was blocked by incubating the cells with mouse serum diluted in FACS buffer (PBS 4% FBS and 2 mM EDTA). Cell surface markers were then stained with antibodies conjugated with fluorochromes (Table 3). Viability was assessed by incubation with Live-Dead Yellow (Invitrogen). The following isotype controls mouse IgG1k BV421, mouse IgG1k PE, mouse IgG1k FITC and mouse IgG1k APC were used for compensation setups. Samples were acquired on LSR Fortessa (BD Bioscience). Analysis of flow cytometric data was performed using FlowJo (TreeStar).Table 3

Antibodies used for flow cytometry

Fluorochrome	Target	Clone	Provider	Ref
BV421	CD90	5E10	Biolegend	328121
PE	CD140a (PDGFRα)	16A1	Biolegend	323505
AF488	CD29	TS2/16	Biolegend	303015
APC	CD105	43A3	Biolegend	323208
APC Vio700	CD45	REA747	Miltenyi	130-110-635
PerCp cy5.5	CD31	WM59	Biolegend	303131

Soussi S., Savchenko L., Rovina D., Iacovoni J.S., Gottinger A., Vialettes M., Pioner J.M., Farini A., Mallia S., Rabino M., Pompilio G., Parini A., Lairez O., Gowran A, & Pizzinat N. (2023). IPSC derived cardiac fibroblasts of DMD patients show compromised actin microfilaments, metabolic shift and pro-fibrotic phenotype. Biology Direct, 18, 41.

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Phenotypic Characterization of hiPSC-Fibroblasts

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Antibodies used for flow cytometry

Fluorochrome	Target	Clone	Provider	Ref
BV421	CD90	5E10	Biolegend	328121
PE	CD140a (PDGFRα)	16A1	Biolegend	323505
AF488	CD29	TS2/16	Biolegend	303015
APC	CD105	43A3	Biolegend	323208
APC Vio700	CD45	REA747	Miltenyi	130-110-635
PerCp cy5.5	CD31	WM59	Biolegend	303131

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Lymphocyte Characterization and Cytokine Production

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Lymphocytes were identified by forward and side scatter, and cell identity was assessed using fluorochrome-conjugated antibodies anti-CD3 (clone SK7; eBioscience), anti-CD4⁺ (RPA-T4; BD), anti-CD8 (RPA-T8; BD), anti-CD161 (DX12; BD), and anti-TCR Vα7.2 (3C10, BioLegend). Viable cells were gated using Live/Dead Yellow or Live/Dead Aqua viability dyes (Invitrogen) according to the manufacturer’s instructions. Cells were stained for 20 minutes in the dark at room temperature, washed, and fixed in PBS containing 2% formaldehyde. All samples were acquired on LSRII or LSRFortessa flow cytometers (BD). Cell division was assessed by labeling PBMCs with 5(6)-carboxyfluorescein diacetate succinimidyl ester (CFSE) (Molecular Probes) for 10 minutes at 37°C. Staining was quenched by the addition of FBS for 5 minutes on ice. Cells were then washed and cultured as described.
For detection of intracellular cytokines, cells were stimulated with 50ng/mL soluble anti-CD3 (HIT3a; BD) and 3μg/mL soluble anti-CD28 (CD28.2; BD) for 24 hours at 37°C and 5% CO₂, in the presence of brefeldin A (GolgiPlug; BD) for the final 6 hours of stimulation. After stimulation, cells were washed, stained with viability dye and antibodies to surface antigens, then fixed and permeabilized using the Cytofix/Cytoperm kit (BD) and stained intracellularly with fluorochrome-conjugated antibody to IFNγ (B27, BD).

Freeman M.L., Morris S.R, & Lederman M.M. (2017). CD161 Expression on Mucosa-Associated Invariant T Cells is Reduced in HIV-Infected Subjects Undergoing Antiretroviral Therapy Who Do Not Recover CD4+ T Cells. Pathogens & Immunity, 2(3), 335-351.

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