Live dead fixable dead cell stain kit

Manufactured by Thermo Fisher Scientific

Sourced in United States, United Kingdom

The LIVE/DEAD Fixable Dead Cell Stain Kit is a fluorescent stain used to identify and distinguish between live and dead cells in a sample. The kit contains a dye that selectively stains dead cells, allowing for their detection and quantification through flow cytometry or other fluorescence-based analysis methods.

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Lab products found in correlation

Facsdiva software, by BD (11 mentions) Lsrfortessa, by BD (11 mentions) Facscanto 2, by BD (11 mentions) Cytofix cytoperm kit, by BD (10 mentions) Foxp3 transcription factor staining buffer set, by Thermo Fisher Scientific (10 mentions) Lsrii flow cytometer, by BD (7 mentions) Cytofix cytoperm, by BD (7 mentions) Ionomycin, by Merck Group (6 mentions) Golgistop, by BD (6 mentions) Lsr 2, by BD (6 mentions) Facscalibur, by BD (6 mentions) Lsrfortessa x 20, by BD (6 mentions) Facscanto 2 flow cytometer, by BD (5 mentions) Fixation permeabilization solution kit, by BD (5 mentions) Golgiplug, by BD (5 mentions) Permeabilization buffer, by Thermo Fisher Scientific (5 mentions) Phorbol myristate acetate (pma), by Merck Group (5 mentions) Facscanto, by BD (5 mentions) Brefeldin a, by Thermo Fisher Scientific (5 mentions) Facsverse, by BD (5 mentions)

239 protocols using live dead fixable dead cell stain kit

Quantifying MSC Viability by Flow Cytometry

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MSC viability was examined by flow cytometry analysis using LIVE/DEAD® Fixable Dead Cell Stain Kit, which was purchased from Life Technologies (Thermo Fisher Scientific, Waltham, MA, USA). Briefly, 1 × 10⁵ MSCs were harvested with 0.25% Trypsin/EDTA, washed with PBS twice, and then incubated with 1 μL of DMSO-diluted stain (Cat. No. L23101) in 1 mL protein-free buffer at 4 °C for 30 minutes. Then the cells were washed in PBS three times and resuspended in a volume of 300 μL PBS. Fluorescence of the cells was detected by flow cytometer (FACS Caliber, BD).

Xue R., Qian Y., Li L., Yao G., Yang L, & Sun Y. (2017). Polycaprolactone nanofiber scaffold enhances the osteogenic differentiation potency of various human tissue-derived mesenchymal stem cells. Stem Cell Research & Therapy, 8, 148.

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AhR Knockdown in HIV-1 Infected CD4+ T Cells

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AhR RNA interference were performed in primary CD4⁺ T cells, as described previously.^{86 (link)} Briefly, memory CD4⁺ T-cells were stimulated by CD3/CD28 Abs for 2 days. Activated cells were nucleofected with 100 μM AhR or non-targeting (NT1) siRNA (ON-TARGETplus SMART pool, Dharmacon) using the Amaxa Human T cell Nucleofector Kit (Lonza, Walkersville, MD, USA), according to the manufacturer’s protocol. Nucleofected cells (2 × 10⁶) were cultured for 24 h at 37°C in the presence of IL-2 (5 ng/mL). Cells were exposed to HIV-1 and cultured up to 3 days. To check the efficacy of KO, the AhR gene expression level was assessed by SYBR Green real-time RT-PCR 24h post-nucleofection. At day 3 post-infection, cells were stained with LIVE/DEAD Fixable Dead Cell Stain Kit (Vivid, Life Technologies,CA) were analyzed by FACS (BD LSRII) (Table S3; key resources table).

Freeman S., Shabi E, & Katan T. (2000). Characterization of Colletotrichum acutatum Causing Anthracnose of Anemone (Anemone coronaria L.). Applied and Environmental Microbiology, 66(12), 5267-5272.

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Multiparameter Flow Cytometry Panel

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The following antibodies were used for flow cytometry: PE-IL-17A (TC11-18H10.1), FITC-B220 (RA3-6B2), PE-α₄β₇ (DATK32), FITC-CD4 (RM4-5), PE/Cy7-CD38 (90), APC-CD138 (281-2), and PE/Cy7-PD-1 (29F.1A12) were purchased from Biolegend; APC-RORγt (B2D), APC-GL-7, and eFluor450-Streptavidin were from eBioscience; Brilliant Violet 421-CD95 (Jo2) and biotinylated CXCR5 (2G8) were from BD Biosciences; recombinant mouse IL-21R human FC chimera was from R&D Systems; AlexaFluor 647 antihuman IgG FCγ (polyclonal) was from Jackson ImmunoResearch; PE-IgA (polyclonal) was from Southern Biotechnology. Intracellular permeabilization was performed with Foxp3 perm/fix kit from eBioscience. Live cells were gated using Live/Dead Fixable Dead Cell stain kit from Life Technologies. Mouse recombinant IL-6, and human recombinant IL-17A, TGFβ1 were purchased from R&D Systems. Recombinant IL-21 was purchased from eBioscience. Antibody against IL-21r (4A9) was from BioXCell. Antibodies against IgA and IgD were purchased from Kirkegaard & Perry Labs and Southern Biotechnology. Anti-µ was purchased from Jackson ImmunoResearch Laboratories. All-trans-retinoic acid, TGFβ receptor I inhibitor SB505124, and collagenase IV were purchased from Sigma-Aldrich. Anti-biotin microbeads from Miltenyi were used to sort naïve IgD⁺ B cells.

Cao A.T., Yao S., Gong B., Nurieva R.I., Elson C.O, & Cong Y. (2015). Interleukin (IL)-21 promotes intestinal IgA response to microbiota. Mucosal immunology, 8(5), 1072-1082.

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Flow Cytometry Antibody Panel

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The following antibodies were used for flow cytometry: Anti-CD19 and anti-IgA antibodies were purchased from Biolegends (San Diego, CA). The Live/Dead Fixable Dead Cell Stain Kit was purchased from Life Technologies (Carlsbad, CA). RAR inhibitor LE135 from Tocris Bioscience (Ellisville, MO), and acetate and butyrate from Sigma. All-trans-RA was purchased from Sigma-Aldrich (St. Louis, MO), TGFβ from Biolegends (San Diego, CA), anti-µ from Jackson ImmunoResearch Laboratories (West Grove, PA), and CD40L from BioXCell (West Lebanon, NH).

Wu W., Sun M., Chen F., Cao A.T., Liu H., Zhao Y., Huang X., Xiao Y., Yao S., Zhao Q., Liu Z, & Cong Y. (2016). Microbiota metabolite short chain fatty acid acetate promotes intestinal IgA response to microbiota which is mediated by GPR43. Mucosal immunology, 10(4), 946-956.

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Multicolor Flow Cytometry of Immune Cells

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After blocking Fc receptors, cells were incubated with appropriately diluted anti-mouse antibodies, including PE-F4/80 (BM8), PerCP-CD45 (30-F11), and APC-CD11b (M1/70) (Biolegend, San Diego, CA, USA). Appropriate species matched Abs served as isotype control. Acquisition of data was performed using a LSRII (BD Biosciences, Mountain View, CA, USA), and data analysis was conducted using the FlowJo software (Tree Star Inc., Ashland, OR, USA). Only live cells determined using LIVE/DEAD Fixable Dead Cell Stain Kit (Life Technologies, Grand Island, NY, USA) were analyzed.

Yoshimura T., Liu M., Chen X., Li L, & Wang J.M. (2015). Crosstalk between Tumor Cells and Macrophages in Stroma Renders Tumor Cells as the Primary Source of MCP-1/CCL2 in Lewis Lung Carcinoma. Frontiers in Immunology, 6, 332.

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Identifying Regulatory T Cells in Pancreas

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Flow cytometry was performed on a FACSCanto II cytometer (BD Biosciences) using antibodies against Foxp3 (eBioscience, FJK-16s), CD4 (BD Biosciences, RM4-5), and CTLA-4 (eBioscience, UC10-4B9). LIVE/DEAD Fixable Dead Cell Stain Kit (Life Technologies) was used to exclude dead cells. Cells were isolated from the pancreas by digestion with collagenase P. Collected data were analyzed using FlowJo software (Tree Star).

Bengsch F., Knoblock D.M., Liu A., McAllister F, & Beatty G.L. (2017). CTLA-4/CD80 pathway regulates T cell infiltration into pancreatic cancer. Cancer immunology, immunotherapy : CII, 66(12), 1609-1617.

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Multiparameter Flow Cytometry Panel

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Cao A.T., Yao S., Gong B., Nurieva R.I., Elson C.O, & Cong Y. (2015). Interleukin (IL)-21 promotes intestinal IgA response to microbiota. Mucosal immunology, 8(5), 1072-1082.

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Dasatinib modulation of MR1 tetramers

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PBMCs were treated with 50 nM dasatinib (Axon Medchem) for 30 min at room temperature. dasatinib-treated cells were stained with hpMR1^−UI, hpMR1^−EC, or hpMR1^−MS tetramers at the indicated concentrations for 1 hour at room temperature. As a comparison, cells were stained with the MR1/5-OP-RU and MR1/6FP tetramers. Cells were then washed, stained with the LIVE/DEAD Fixable Dead Cell Stain Kit (Life Technologies), and surface-stained with the antibodies listed in table S1 for 30 min at 4°C. Samples were fixed with 1% paraformaldehyde, and acquisition was performed using a Fortessa flow cytometer with FACSDiva software (BD Biosciences). All flow cytometry data were analyzed using FlowJo software (TreeStar) and Prism (GraphPad). Gating strategies are shown in fig. S2.

Harriff M.J., McMurtrey C., Froyd C.A., Jin H., Cansler M., Null M., Worley A., Meermeier E.W., Swarbrick G., Nilsen A., Lewinsohn D.A., Hildebrand W., Adams E.J, & Lewinsohn D.M. (2018). MR1 displays the microbial metabolome driving selective MR1-restricted T cell receptor usage. Science immunology, 3(25), eaao2556.

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Flow Cytometry Analysis of Dead Cells

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Single-cell suspensions were stained with LIVE/DEAD Fixable Dead Cell Stain Kit (Life Technologies) to identify dead cells. Intracellular staining was performed using eBioscience’s FOXP3 staining kit according to the manufacturer’s instructions. Data was acquired on a LSRFortessa flow cytometer (BD Biosciences) and analyzed with FlowJo Version 7.6.4 software for Mac (TreeStar).

Mailer R.K., Joly A.L., Liu S., Elias S., Tegner J, & Andersson J. (2015). IL-1β promotes Th17 differentiation by inducing alternative splicing of FOXP3. Scientific Reports, 5, 14674.

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Organoid Lipid Content Profiling

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Organoids were harvested and dissociated in to single cells by TrypLE Express (Invitrogen) including Y-27632 and N-acetyl-L-cysteine, as described above. Cells were first stained with the LIVE/DEAD Fixable Dead Cell Stain Kit (Life Technologies/Thermo Fisher Scientific). After washing, LipidTox green from HCS LipidTox Phospholipidosis and Steatosis Detection Kit (Thermo Fisher Scientific) was added to samples 30 min before analysis by flow cytometry.

Li S., Lu C.W., Diem E.C., Li W., Guderian M., Lindenberg M., Kruse F., Buettner M., Floess S., Winny M.R., Geffers R., Richnow H.H., Abraham W.R., Grassl G.A, & Lochner M. (2022). Acetyl-CoA-Carboxylase 1-mediated de novo fatty acid synthesis sustains Lgr5+ intestinal stem cell function. Nature Communications, 13, 3998.

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