Live dead fixable dead cell staining kit

Manufactured by Thermo Fisher Scientific

Sourced in United States

The Live/Dead Fixable Dead Cell staining kit is a fluorescent dye-based product designed to identify and quantify dead cells in a sample. It provides a simple and effective way to detect and measure cell viability.

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Lab products found in correlation

Facs cytexpert software, by Beckman Coulter (3 mentions) Cytoflex flow cytometer, by Beckman Coulter (3 mentions) Lsrii flow cytometer, by BD (2 mentions) Lsrfortessa, by BD (2 mentions) Human fc block, by BD (1 mentions) Fixation permeabilization solution kit, by BD (1 mentions) Transcription factor fixation permeabilization buffer, by BD (1 mentions) Facsverse, by BD (1 mentions) Facs lsr 2, by BD (1 mentions) Fixation buffer, by BioLegend (1 mentions) Cell staining buffer, by BioLegend (1 mentions) Intracellular staining permeabilization wash buffer, by BioLegend (1 mentions) True nuclear transcription factor buffer set, by BioLegend (1 mentions) Human trustain fcx, by BioLegend (1 mentions) Facscelesta flow cytometer, by BD (1 mentions) Anti pd 1 clone eh12.2h7, by BioLegend (1 mentions) Anti lag 3, by R&D Systems (1 mentions) Anti foxp3 clone pch101, by Thermo Fisher Scientific (1 mentions) Anti ctla 4 clone bni3, by BD (1 mentions) Foxp3 staining buffer, by Thermo Fisher Scientific (1 mentions)

Close spelling variants of this product

Fixable live/dead Live/Dead staining

11 protocols using live dead fixable dead cell staining kit

Multicolor Flow Cytometry Analysis of NK Cells

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Cultured primary NK cells and NK-92 cells were stained with the LIVE/DEAD Fixable Dead Cell Staining Kit (ThermoFisher, Waltham, MA) and anti-CD56 (HCD56), anti-CD3 (OKT3), anti-KIR2DL1 (HP-MA4), anti-KIR2DL2/L3 (DX27), anti-KIR3DL1 (DX9), and anti-CD94 (DX22) flurochrome-conjugated antibodies (all from Biolegend) and analyzed on an LSR II flow cytometer (BD Biosciences). Data was analyzed using FlowJo (v10.6.0) software (BD Biosciences). For all flow cytometry experiments, lymphocytes were first gated as determined by size in forward/side scatter plots. NK cells were then gated as CD3⁻CD56⁺. For transduction experiments, cells were further gated as GFP⁺, mCherry⁺, or blue⁺.

Wu C.Y., Zhang B., Kim H., Anderson S.K., Miller J.S, & Cichocki F. (2020). Ascorbic Acid Promotes KIR Demethylation During Early NK Cell Differentiation. Journal of immunology (Baltimore, Md. : 1950), 205(6), 1513-1523.

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Single-cell Immunophenotyping Protocol

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Single-cell suspensions were immunostained with a panel of fluorochrome-tagged monoclonal antibodies (online supplemental table 3). Red cells were lysed with an ammonium chloride solution, and samples were incubated with Live/Dead Fixable Dead Cell Staining Kit (ThermoFisher) before staining. Samples stained with isotype-matched antibodies were used as negative controls. For sample acquisition, a Beckman Coulter cytoflex flow cytometer with FACS CytExpert software was used (Beckman Coulter), and FlowJo software (Tree Star) was used for analyses.

Yang M., Lu J., Zhang G., Wang Y., He M., Xu Q., Xu C, & Liu H. (2021). CXCL13 shapes immunoactive tumor microenvironment and enhances the efficacy of PD-1 checkpoint blockade in high-grade serous ovarian cancer. Journal for Immunotherapy of Cancer, 9(1), e001136.

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Single-cell Flow Cytometry Analysis

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Single‐cell suspensions were stained with a panel of fluorochrome‐tagged antibodies (Table S2). Samples were incubated with Live/Dead Fixable Dead Cell Staining Kit (ThermoFisher) and human BD Fc Block (BD Biosciences), then stained with the indicated monoclonal antibodies (mAbs) for 30 min at 4°C in dark. When necessary, before staining with antibodies against intracellular proteins or transcription factors, cells were pretreated with Fixation/Permeabilization Solution Kit or Transcription Factor Fixation/Permeabilization buffer (BD Biosciences), respectively, according to the manufacturer's instructions. For sample acquisition, a Beckman Coulter cytoflex flow cytometer with FACS CytExpert software was used (Beckman Coulter), and FlowJo software (Tree Star) was used for analyses.

Wang Y., He M., Zhang G., Cao K., Yang M., Zhang H, & Liu H. (2021). The immune landscape during the tumorigenesis of cervical cancer. Cancer Medicine, 10(7), 2380-2395.

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Cell Binding Assay with F7AK3

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Tumors cells were collected, washed with PBS, and incubated with FACS buffer (1×PBS with 0.5% BSA) containing various concentrations of F7AK3 for 30 min at room temperature. Subsequently cells were washed with FACS buffer twice and stained with PE-conjugated goat-anti human IgG Fc antibody (Invitrogen, 2234986). Dead cells were identified using Live/Dead Fixable Dead Cell staining kit (Invitrogen, 2123595). Samples were analyzed by flow cytometry with a FACS LSR II or FACS Verse (BD).

Liu H., Bai L., Huang L., Ning N., Li L., Li Y., Dong X., Du Q., Xia M., Chen Y., Zhao L., Li Y., Meng Q., Wang J., Duan Y., Ming J., Yuan A.Q, & Yang X.P. (2021). Bispecific antibody targeting TROP2xCD3 suppresses tumor growth of triple negative breast cancer. Journal for Immunotherapy of Cancer, 9(10), e003468.

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Immunophenotyping of human and murine cells

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Cell suspensions from human EOC tissues or ascites, and peripheral blood of HDs or patients with EOC, and subcutaneous tumours of B6C3F1 mice were stained with a panel of fluorochrome-tagged antibodies (Supplementary Table 3). Red cells were lysed with an ammonium chloride solution and samples were incubated with Live/Dead Fixable Dead Cell Staining Kit (Invitrogen) prior to staining to permit the identification of live cells. Samples stained with isotype-matched antibodies were used as negative controls. For sample acquisition, a Beckman Coulter cytoflex flow cytometer with FACS CytExpert software (Beckman Coulter) was used, and FlowJo software (Tree Star) used for the analyses.

Yang M., Zhang G., Wang Y., He M., Xu Q., Lu J., Liu H, & Xu C. (2020). Tumour-associated neutrophils orchestrate intratumoural IL-8-driven immune evasion through Jagged2 activation in ovarian cancer. British Journal of Cancer, 123(9), 1404-1416.

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Multiparametric Flow Cytometry of Cervical Immune Cells

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Multiparametric flow cytometry was used to assess the expression of activation markers (CD38+ or HLA‐DR+), the maker of proliferation (Ki67+) and the HIV co‐receptor (CCR5+) on CD4+ T cells in cervical cytobrush‐derived specimens. The viability of cervical mononuclear cells (CMCs) was determined using the LIVE/DEAD™ Fixable Dead Cell Staining Kit (Invitrogen Life Technologies, Carlsbad, CA, USA) as outlined in the manufacturer's protocol. CMCs were treated with antibody‐conjugated fluorophores, washed and fixed (Table S2). Data were acquired on an LSRII flow cytometer (BD Immunocytometry Systems, San Jose, CA, USA) and analysed with FlowJo™ Software version 9.9 (Tree Star, Inc., US). The gating strategy was previously published [24].

Jewanraj J., Ngcapu S., Osman F., Ramsuran V., Fish M., Mtshali A., Singh R., Mansoor L.E., Abdool Karim S.S., Abdool Karim Q., Passmore J.S, & Liebenberg L.J. (2021). Transient association between semen exposure and biomarkers of genital inflammation in South African women at risk of HIV infection. Journal of the International AIDS Society, 24(6), e25766.

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Single-Cell Profiling of Bladder Cancer Samples

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All 32 fresh human specimens were obtained from patients underwent radical cystectomy in Shanghai Cancer Center, Zhongshan Hospital, and Shanghai General Hospital, respectively. Single cells were isolated from freshly resected tumor tissues using collagenase IV. About 1,000,000 cells were collected and incubated in RBC lysis buffer at 4°C for 5 min. Surface markers (listed in supplementary table 2) were stained in Cell Staining Buffer for 30 min at 4°C in dark after blocking Fc-receptors with Human TruStain FcX (Biolegend). Then, cells were fixed by Fixation Buffer (Biolegend), permeabilized by Intracellular staining permeabilization wash buffer (Biolegend) and intracellular cytokine (listed in supplementary table 2) was stained for 40 min at 4°C in dark. Specially, we performed transcription factor staining using the True-Nuclear transcription Factor Buffer set (Biolegend) according to the manufacturer’s instructions. Dead cells were excluded by Live/Dead Fixable Dead Cell StainingKit (Invitrogen). Cells were acquired on the FACSCelesta Flow Cytometer (BD Biosciences) and analyzed with FlowJo software (Treestar).

Wang Z., Zhou Q., Zeng H., Zhang H., Liu Z., Huang Q., Xiong Y., Wang J., Chang Y., Bai Q., Xia Y., Wang Y., Zhu Y., Xu L., Dai B., Liu L., Guo J, & Xu J. (2020). Tumor-infiltrating IL-17A+ cells determine favorable prognosis and adjuvant chemotherapeutic response in muscle-invasive bladder cancer. Oncoimmunology, 9(1), 1747332.

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Comprehensive Immune Cell Profiling

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The following monoclonal antibodies and reagents were used: for surface staining, anti-CD25 (clone M-A251), anti-CD45RA (clone 5H9), anti-CXCR3 (clone 1C6/XCXR3), anti-CCR6 (clone 11A9) and anti-CTLA-4 (clone BNI3) from BD Biosciences, anti-CD127 (clone 17-1278) from eBioscience, anti-TIM-3 (clones F38-2E2 and 344823 from Biolegend and R&D systems respectively), anti-PD-1 (clone EH12.2H7) from Biolegend, anti-LAG-3 (polyclonal) from R&D systems and Live/Dead fixable dead cell staining kit from Invitrogen; for intracellular staining, anti-FoxP3 (clone PCH101) from eBioscience and anti-pSTAT3 (pY705) from BD Biosciences.

Gautron A.S., Dominguez-Villar M., de Marcken M, & Hafler D.A. (2014). Enhanced Suppressor Function of TIM-3+ FoxP3+ Regulatory T cells. European journal of immunology, 44(9), 2703-2711.

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Treg Suppression of Effector T Cell Proliferation

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Tregs were co-cultured with CFSE-labeled Tresp, Th1 or Th17 at 1:2, 1:4 and 1:8 Treg:Tresp ratios (2000 Tregs). Treg inspector beads (Miltenyi) were used as a stimulus at a 1:2 cell:bead ratio. At day 3, co-cultures were stained for viability using the Live/Dead fixable dead cell staining kit (Invitrogen) and fixed using the FoxP3 staining buffer (eBioscience). Proliferation of viable responder T cells was analyzed by CFSE dilution on a BD LSR Fortessa (BD Biosciences).

Gautron A.S., Dominguez-Villar M., de Marcken M, & Hafler D.A. (2014). Enhanced Suppressor Function of TIM-3+ FoxP3+ Regulatory T cells. European journal of immunology, 44(9), 2703-2711.

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Dissociation and Immunophenotyping of Lung Tumors

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To generate cell suspensions, tumor nodules were excised from lungs of KP and TL mice, cut into small pieces, and further dissociated in RPMI-1640 buffer containing 5% FBS, 100 IU/ml collagenase type IV (Invitrogen, Carlsbad, CA), and 50 μg/ml DNAse I (Roche, Basel, Switzerland) for 45 min at 37°C. After incubation, cells were treated with red blood cell lysis buffer and filtered through a 70 μm cell strainer. After centrifugation, cell pellets were resuspended in 1X PBS/2% FBS. About 0.5–1 × 10⁶ cells were stained for surface markers in 1X PBS/2% FBS for 15 min at 4°C. Intracellular staining was performed for Foxp3 using the Foxp3 staining kit (ebioscience, Santa Clara, CA) according to manufacturer’s instructions. The Cytofix/Cytoperm kit (BD Biosciences, San Jose, CA) was used for intracellular cytokine staining. Briefly, fixed and permeabilized cells were stained for surface markers, including CD4, CD8, and CD3, followed by intracellular staining with PE-conjugated anti-IFN-γ, or isotype-matched mAbs. In all stained samples, dead cells were excluded using Live/Dead Fixable Dead Cell staining kit (Invitrogen, Carlsbad, CA). Cells were acquired on the LSR Fortessa (BD Biosciences) and analyzed with FlowJo software (Treestar). Gating strategy for FACS analyses are shown in Supplementary Figure 24.

Adeegbe D.O., Liu Y., Lizotte P.H., Kamihara Y., Aref A.R., Almonte C., Dries R., Li Y., Liu S., Wang X., Warner-Hatten T., Castrillon J., Yuan G.C., Poudel-Neupane N., Zhang H., Guerriero J.L., Han S., Awad M.M., Barbie D.A., Ritz J., Jones S.S., Hammerman P.S., Bradner J., Quayle S.N, & Wong K.K. (2017). Synergistic immunostimulatory effects and therapeutic benefit of combined histone deacetylase and bromodomain inhibition in non-small cell lung cancer. Cancer discovery, 7(8), 852-867.

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