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Live dead fixable yellow dead cell stain

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The LIVE/DEAD Fixable Yellow Dead Cell Stain is a fluorescent dye that binds to dead cells, allowing for their identification and exclusion from analysis. The stain can be used with a variety of flow cytometry and fluorescence microscopy applications.

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Lab products found in correlation

Lsrii flow cytometer, by BD (5 mentions) Cd3 apc cyanine7, by BioLegend (4 mentions) Cd8a fitc, by BioLegend (4 mentions) Lsrfortessa, by BD (4 mentions) Fixation permeabilization buffer, by Thermo Fisher Scientific (4 mentions) Foxp3 staining kit, by Thermo Fisher Scientific (3 mentions) Lsrii flow cytometer, by Tree Star (2 mentions) Cd107b alexa fluor 647, by BioLegend (2 mentions) Brilliant violet 711, by BioLegend (2 mentions) Foxp3 efluor450, by Thermo Fisher Scientific (2 mentions) Cd25 pe cy5, by BioLegend (2 mentions) Cd279 pd 1 pe, by BioLegend (2 mentions) Tnf α brilliant violet 421, by BioLegend (2 mentions) Il 2 brilliant violet 711, by BioLegend (2 mentions) Ifn brilliant violet 785, by BioLegend (2 mentions) 7 aad, by BD (2 mentions) 4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions) Monensin, by BioLegend (2 mentions) Aria 2 cell sorter, by BD (2 mentions) Brdu flow kit, by BD (2 mentions)

Spelling variants of this product

LIVE/DEAD stain (166 citations) Fixable live/dead stain (61 citations) Live/Dead Fixable Yellow (38 citations) Live/Dead Yellow (37 citations) Live/Dead Yellow stain (5 citations) Fixable live/dead (2 citations)

51 protocols using live dead fixable yellow dead cell stain

1

Flow Cytometry Analysis of Antigen-Specific CD8+ T Cells

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Spleen cell suspensions prepared from immunized mice were adjusted to 1 × 106 cells/mL and incubated with LIVE/DEAD® Fixable Yellow Dead Cell Stain (Life Technologies / Thermo Fisher) diluted 1:1000 in PBS for 30 min at 4 °C. After washing with PBS, cells were incubated with APC-labeled H2-Db dextramers (Immudex, Copenhagen, Denmark) loaded with NY-BR-11241-1249 or with Human Papilloma Virus (HPV) 16 E749–57, diluted as indicated in 60 μL FACS buffer for 30 min at room temperature in the dark. Fluorochrome-labeled antibodies against CD3 (clone 17A2), CD8 (clone 53–6.7), CD4 (clone GK1.5) CD14 (clone Sa14–2) (all purchased from Biolegend, San Diego, CA), and the respective isotype controls were diluted 1:50 in 60 μL FACS buffer and added to the cells without washing off the dextramers so that the final dilution of the antibodies was 100-fold. Cells were incubated with the antibody mix at 4 °C for 30 min. The cells were then washed and analyzed by flow cytometry for viable CD3+CD14−CD8+ dextramer+ cells.
Das K., Eisel D., Vormehr M., Müller-Decker K., Hommertgen A., Jäger D., Zörnig I., Feuerer M., Kopp-Schneider A., Osen W, & Eichmüller S.B. (2019). A transplantable tumor model allowing investigation of NY-BR-1-specific T cell responses in HLA-DRB1*0401 transgenic mice. BMC Cancer, 19, 914.
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2

Multiparametric Phenotyping of T Cell Subsets

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To define the memory vs. naïve subsets, the following antibodies were used: SP34–2 (CD3; Alexa700, BD Biosciences), L200 (CD4; AmCyan, BD Bioscienes), SK-1 (CD8; PerCP-cy-5.5, BD Biosciences), MAB11 (TNFα; FITC, eBioscience), B27 (IFNγ; APC, BD Pharmingen), FN50 (CD69; PE, BD Biosciences), CD28.2 (CD28; PE-TexasRed, BD Biosciences), 150503 (CCR7; R&D Systems), and SA (PacBlue, BD Biosciences). CCR7 was biotinylated using the EZ-link Malemide-PEO Solid Phase Biotinylation kit from Fisher Scientific, as per manufacturer instructions. For cell recognition and T cell response assays, the following antibodies were used: anti-CD3 (clone: SP34–2, Pacific Blue; BD Biosciences), anti-CD8 (clone: SK1, TruRed; BD Biosciences), anti-CD4 (clone: L200, PE-Cy7, BV395; BD Biosciences), anti-CD28 (clone: CD28.2, PE; BD Biosciences), anti-CD95 (clone: DX2, FITC; BD Biosciences), anti-CD69 (clone: FN50, ECD, Biolegend), anti-CCR7 (clone: 150503, Pacific Blue, R&D Systems), anti-IFNγ (clone: B27, FITC; BD Biosciences), anti-TNFα (clone: MAb11, Alexa 700; BD Biosciences), and LIVE/DEAD Fixable Yellow Dead Cell Stain (Life Technologies) was used to assess cell viability.
Malouli D., Gilbride R.M., Wu H.L., Hwang J.M., Maier N., Hughes C.M., Newhouse D., Morrow D., Ventura A.B., Law L., Tisoncik-Go J., Whitmore L., Smith E., Golez I., Chang J., Reed J.S., Waytashek C., Weber W., Taher H., Uebelhoer L.S., Womack J.L., McArdle M.R., Gao J., Papen C.R., Lifson J.D., Burwitz B.J., Axthelm M.K., Smedley J., Früh K., Gale M J.r., Picker L.J., Hansen S.G, & Sacha J.B. (2022). Cytomegalovirus Vaccine-induced Unconventional T cell Priming and Control of SIV Replication is Conserved Between Primate Species. Cell host & microbe, 30(9), 1207-1218.e7.
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3

Identifying Immune Cells via Dextramers

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Fc receptors on PBMCs or splenocytes (0.5–1 × 106) were blocked by incubating with a human FcR blocking reagent (Miltenyi Biotec, Woking, UK, Catalogue number 130-059-901) or a murine CD16/CD32 mAb (BioLegend, London, UK clone 93) for 15 min at 4 °C, after which they were incubated with 16–32 × 10−9 M of the appropriate MHC class I Dextramer™ (Immudex, Copenhagen, Denmark) for 10 min at 4 °C in the dark. PBMCs were then incubated with human mAbs for CD8, CD3, and CD19 (Table A2) and splenocytes with murine mAbs for CD8, CD3, PD-1, Tim-3, and LAG-3 for 30 min at 4 °C in the dark. LIVE/DEAD™ Fixable Yellow Dead Cell Stain (Life Technologies, ThermoFisher Scientific, Paisley, UK) identified viable cells. Samples were acquired and analyzed as described in Section 2.3.
Vu P.L., Vadakekolathu J., Idri S., Nicholls H., Cavaignac M., Reeder S., Khan M.A., Christensen D., Pockley A.G, & McArdle S.E. (2022). A Mutated Prostatic Acid Phosphatase (PAP) Peptide-Based Vaccine Induces PAP-Specific CD8+ T Cells with Ex Vivo Cytotoxic Capacities in HHDII/DR1 Transgenic Mice. Cancers, 14(8), 1970.
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4

Multiparameter Flow Cytometry of Immune Cells

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Fluorochrome-conjugated anti-mouse or -human monoclonal antibodies included anti-CD3, CD4, CD8, CD25, CD25RO, CD83, CD86, HLA-DR, CD127, CCR6, CCR7, Ki-67, Foxp3, IFNγ, IL-4, IL-17A, XBP-1s, and phosphorylated STAT3 Y705 (Supplemental Table 1). Live/Dead Fixable Yellow Dead Cell Stain (Life Technologies) was used to determine viability. Live events were acquired on a FACSCanto (FlowJo software, ver. 7.6.4).
Betts B.C., Locke F.L., Sagatys E.M., Pidala J., Walton K., Menges M., Reff J., Saha A., Djeu J.Y., Kiluk J.V., Lee M.C., Kim J., Kang C.W., Tang C.H., Frieling J., Lynch C.C., List A., Rodriguez P.C., Blazar B.R., Conejo-Garcia J.R., Del Valle J.R., Hu C.C, & Anasetti C. (2018). Inhibition of Human Dendritic Cell ER Stress Response Reduces T Cell Alloreactivity Yet Spares Donor Anti-tumor Immunity. Frontiers in Immunology, 9, 2887.
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5

Comprehensive Immune Cell Profiling

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Fluorochrome-conjugated mouse anti-human monoclonal antibodies included anti-CD3, CD4, CD8, CD25, CD39, CD127, CTLA4, Foxp3, LAG3, pSTAT3 Y705, pSTAT5 Y694, and pH3Ser10 (BD Biosciences, eBioscience, and Cell Signaling Technology). LIVE/DEAD Fixable Yellow Dead Cell Stain (Life Technologies) was used to determine viability. Live events were acquired on a FACSCanto or LSR II Flow Cytometer (FlowJo software, version 7.6.4; Tree Star).
Betts B.C., Veerapathran A., Pidala J., Yang H., Horna P., Walton K., Cubitt C.L., Gunawan S., Lawrence H.R., Lawrence N.J., Sebti S.M, & Anasetti C. (2017). Targeting Aurora kinase A and JAK2 prevents GVHD while maintaining Treg and antitumor CTL function. Science translational medicine, 9(372), eaai8269.
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6

Multiparameter Flow Cytometry Immunophenotyping

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The following conjugated antibodies were used in these studies: a) from BD Biosciences, SP34-2 (CD3; PacBlu, Alexa700), SK1 (CD8a; TruRed, AmCyan), 25723.11 (IFN-γ; APC), MAb11 (TNF; FITC, Alexa700), DX2 (CD95; FITC), 28.2 (CD28; PE), 2H7 (CD20; APC-H7), RPA-T8 (CD8; PacBlu), L200 (CD4; PerCP-Cy5.5), 3G8 (CD16; FITC), b) from Biolegend, W6/32 (pan-MHC-I; PerCP-Cy5.5), OKT-4 (CD4; PE-Cy7), c) from Miltenyi Biotec, M-T466 (CD4; APC), d) from eBiosciences, M1-14D12 (mouse IgG1; PE-Cy7), e) from Life Technologies, Goat-Anti-Mouse (H+L) F(ab′)2 fragment (APC), f) from Beckman Coulter, RMO52 (CD14; ECD), and g) from the Nonhuman Primate Reagent Resource, Mamu-A1*001:01-PE (cat. no. PR-1102). The following unconjugated antibodies were used in these studies: a) from Advanced BioScience Laboratories, 4324 (SIV Gag p27), conjugated in-house to FITC using FluoReporter FITC Protein Labeling Kit (Invitrogen), b) 4D12 (HLA-E), grown and purified in-house, and c) W6/32 (pan-MHC-I), grown and purified in-house. Live/dead Fixable Yellow Dead Cell Stain (Life Technologies) was used to assess cell viability.
Wu H.L., Wiseman R.W., Hughes C.M., Webb G.M., Abdulhaqq S.A., Bimber B.N., Hammond K.B., Reed J.S., Gao L., Burwitz B.J., Greene J.M., Ferrer F., Legasse A.W., Axthelm M.K., Park B.S., Brackenridge S., Maness N.J., McMichael A.J., Picker L.J., O’Connor D.H., Hansen S.G, & Sacha J.B. (2017). The role of MHC-E in T cell immunity is conserved among humans, rhesus macaques, and cynomolgus macaques. Journal of immunology (Baltimore, Md. : 1950), 200(1), 49-60.
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7

Multi-parameter Immune Cell Profiling

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CD44‐Brilliant Violet 711 (BioLegend, Cat.# 103057, clone: IM7, 1:80), CD8a‐FITC (BioLegend, Cat.# 100706, clone: 53‐6.7, 1:50), CD62L‐PE (BioLegend, Cat.# 104407, clone: MEL‐14, 1:80), CX3CR1‐PE/Cyanine 7 (BioLegend, Cat.# 149015, clone: SA011F11, 1:1333), CD107b‐Alexa Fluor 647 (BioLegend, Cat.# 108512, clone: M3/84, 1:200), CD3‐APC/Cyanine7 (BioLegend, Cat.# 100222, clone: 17A2, 1:80), and LIVE/DEAD Fixable Yellow Dead Cell Stain (Thermo Fisher Scientific, Cat.# L34967, 1:7500).
Matos A.I., Peres C., Carreira B., Moura L.I., Acúrcio R.C., Vogel T., Wegener E., Ribeiro F., Afonso M.B., Santos F.M., Martínez‐Barriocanal Á., Arango D., Viana A.S., Góis P.M., Silva L.C., Rodrigues C.M., Graca L., Jordan R., Satchi‐Fainaro R, & Florindo H.F. (2023). Polyoxazoline‐Based Nanovaccine Synergizes with Tumor‐Associated Macrophage Targeting and Anti‐PD‐1 Immunotherapy against Solid Tumors. Advanced Science, 10(25), 2300299.
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8

Characterization of Tfh Cell Cytokines

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Sorted tonsil or FL R5-PD-1dim and GC-Tfh were cultured for 3 days in 10% FCS-RPMI 1640 with pre-seeded TSCs or FRCLs (5:1 ratio) in presence of anti-CD3 (0.2 ug/ml) and anti-CD28 (0.2 ug/ml) stimulating antibodies. After 3 days, a restimulation step was done with 100 ng/ml phorbol myristate acetate and 750 ng/ml ionomycin for 6 h, supplemented with GolgiPlug (Becton Dickinson) for the last 4 h. For inhibition experiments, Notch chemical inhibitor L685,458 (Sigma Aldrich) or blocking antibodies (bAbs) (Supplemental Table 1) were used. The percentage of singlet viable T cells producing IL-4, IL-21, and IFN-γ was determined by staining with live/dead fixable yellow dead cell stain (Thermo Fisher Scientific) and CD2, followed by fixation in paraformaldehyde 4% for 15min, permeabilization with saponin 0.5%, and staining for intracellular cytokines.
Misiak J., Jean R., Rodriguez S., Deleurme L., Lamy T., Tarte K, & Amé-Thomas P. (2020). Human Lymphoid Stromal Cells Contribute to Polarization of Follicular T Cells Into IL-4 Secreting Cells. Frontiers in Immunology, 11, 559866.
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9

Flow Cytometry Characterization of Immune Cells

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MHC Class II‐VioBlue (Miltenyi Biotec, Cat.# 130‐112‐237, clone: REA813, 1:50), CD206 (mannose receptor)‐Brilliant Violet 711 (BioLegend, Cat.# 141727, clone: C068C2, 1:40), CD11b‐Vio Bright FITC (Miltenyi Biotec, Cat.# 130‐113‐805, clone: REA592, 1:50), CD45‐PerCP‐Vio 700 (Miltenyi Biotec, Cat.# 130‐110‐663/130‐110‐801, clone: REA737, 1:50), F4/80‐PE‐Vio 770 (Miltenyi Biotec, Cat.# 130‐118‐459, clone: REA126, 1:50), Gr1‐APC (Miltenyi Biotec, Cat.# 130‐112‐307, clone: REA810, 1:50), and LIVE/DEAD Fixable Yellow Dead Cell Stain (Thermo Fisher Scientific, Cat.# L34967, 1:1000).
Matos A.I., Peres C., Carreira B., Moura L.I., Acúrcio R.C., Vogel T., Wegener E., Ribeiro F., Afonso M.B., Santos F.M., Martínez‐Barriocanal Á., Arango D., Viana A.S., Góis P.M., Silva L.C., Rodrigues C.M., Graca L., Jordan R., Satchi‐Fainaro R, & Florindo H.F. (2023). Polyoxazoline‐Based Nanovaccine Synergizes with Tumor‐Associated Macrophage Targeting and Anti‐PD‐1 Immunotherapy against Solid Tumors. Advanced Science, 10(25), 2300299.
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10

Multiparametric Flow Cytometry Analysis of T-Cell Subsets

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FOXP3‐eFluor 450 (eBioscience, Cat.# 48‐5773‐82, clone: FJK‐16s, 1:333), CD95 (Fas)‐Brilliant Violet 605 (BioLegend, Cat.# 152612, clone: SA367H8, 1:80), CD185 (CXCR5)‐Brilliant Violet 711 (BioLegend, Cat.# 145529, clone: L138D7, 1:40), CD4‐PerCP/Cyanine5.5 (BioLegend, Cat.# 100434, clone: GK1.5, 1:80), CD279 (PD‐1)‐PE (BioLegend, Cat.# 135206, clone: 29F.1A12, 1:20), CD25‐PE/Cy5 (BioLegend, Cat.# 102010, clone: PC61, 1:80), GL7‐eFluor 660 (eBioscience, Cat.# 50‐5902‐82, clone: GL7, 1:80), CD19‐APC/Cyanine7 (BioLegend, Cat.# 115530, clone: 6D5, 1:20), and LIVE/DEAD Fixable Yellow Dead Cell Stain (Thermo Fisher Scientific, Cat.# L34967, 1:7500).
Matos A.I., Peres C., Carreira B., Moura L.I., Acúrcio R.C., Vogel T., Wegener E., Ribeiro F., Afonso M.B., Santos F.M., Martínez‐Barriocanal Á., Arango D., Viana A.S., Góis P.M., Silva L.C., Rodrigues C.M., Graca L., Jordan R., Satchi‐Fainaro R, & Florindo H.F. (2023). Polyoxazoline‐Based Nanovaccine Synergizes with Tumor‐Associated Macrophage Targeting and Anti‐PD‐1 Immunotherapy against Solid Tumors. Advanced Science, 10(25), 2300299.
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