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Live dead fixable near ir stain kit

Manufactured by Thermo Fisher Scientific
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The LIVE/DEAD® fixable Near-IR stain kit is a fluorescent dye that can be used to detect cell viability. It binds to proteins in both live and dead cells, emitting near-infrared fluorescence. This kit is designed for use in flow cytometry and other fluorescence-based applications.

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Lab products found in correlation

Facsaria 3, by BD (4 mentions) Ionomycin, by Merck Group (3 mentions) Foxp3 staining kit, by Thermo Fisher Scientific (3 mentions) Golgistop, by BD (3 mentions) Ki67 16a8, by Thermo Fisher Scientific (2 mentions) Cyan adp, by Beckman Coulter (2 mentions) Cytoflex flow cytometer, by Beckman Coulter (2 mentions) Sytox blue, by Thermo Fisher Scientific (1 mentions) Phorbol 12 myristate 13 acetate, by Merck Group (1 mentions) 4 6 diamidine 2 phenylindole dihydrochloride, by Merck Group (1 mentions) Lsrfortessa flow cytometer, by BD (1 mentions) Aurora spectral analyzer, by Cytek (1 mentions) Facs aria 3 machine, by BD (1 mentions) Tcl buffer, by Qiagen (1 mentions) Cyan adp flow cytometer, by Beckman Coulter (1 mentions) Klrg1 2f1, by BioLegend (1 mentions) Lsrii fortessa, by BD (1 mentions) 647 edu click proliferation kit, by BD (1 mentions) Complete keratinocyte growth medium 2, by PromoCell (1 mentions) Facsdiva, by BD (1 mentions)

Close spelling variants of this product

LIVE/DEAD fixable near-infrared (IR) dead cell stain kit LIVE/DEAD Fixable Near-IR Dead Cell Stain Kit LIVE/DEAD Fixable Violet or Near-IR Dead Cell Stain kit LIVE/DEAD Fixable Near-IR cell stain kit LIVE-DEAD® Fixable yellow or near-IR dead cell stain kit Live/Dead fixable near-IR stain LIVE/DEAD Fixable Near-IR LIVE/DEAD Near IR stain Live/Dead Near-IR Fixable live/dead stain

9 protocols using live dead fixable near ir stain kit

1

Multiparametric Flow Cytometry Analysis

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Cell suspensions from lymphoid organs were stained with fluorochrome-conjugated anti-mouse CD3 (17A2), CD4 (RM4-5), CD8α (53-6.7), CD11c (N418), CD19 (6D5), CD25 (PC61.5), CD40 (1C10), CD44 (IM7), CD45 (30-F11), CD45.1 (A20), CD49b (DX5), CD62L (MEL-14), CD70 (FR70), CD83 (Michel-17), CD86 (GL1), CCR7 (4B12), CXCR3 (CXCR3-173), EpCAM (G8.8), F4/80 (BM8), Ly51 (6C3), Ly51 (FG35.4), CD90.1 (HIS51), CD90.2 (53-2.1), CD137L (TKS-1), CD252 (RM134L), TCRβ (H57-597) and anti-human CD2 (RPA-2.10) that were purchased from either BD Biosciences, Biolegend or eBioscience. Intracellular staining was performed with Foxp3 staining kit for Foxp3 (FJK-16s) and IFN-γ (XMG1.2) Abs according to manufacturer's instructions (eBioscience). For IFN-γ cytokine staining, cells were stimulated with phorbol 12-myristate 13-acetate (Sigma) and ionomycin (Sigma) for 4 h and with brefeldin A for the last 2 h. For dead cell exclusion LIVE/DEAD® fixable Near-IR stain kit (Invitrogen) or Sytox Blue (Molecular Probes) was used. Flow cytometric analysis was performed on LSR II (BD Biosciences) and data were analyzed using FlowJo software (Tree Star).
Garg G., Nikolouli E., Hardtke-Wolenski M., Toker A., Ohkura N., Beckstette M., Miyao T., Geffers R., Floess S., Gerdes N., Lutgens E., Osterloh A., Hori S., Sakaguchi S., Jaeckel E, & Huehn J. (2017). Unique properties of thymic antigen-presenting cells promote epigenetic imprinting of alloantigen-specific regulatory T cells. Oncotarget, 8(22), 35542-35557.
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2

Comprehensive Immune Cell Phenotyping

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Cell suspensions were treated with Fc block CD16/CD32 (2.4G2) and surface markers were stained with fluorochrome conjugated anti-mouse CD3 (145-2C11), CD4 (RM4-5), CD11b (M1/70), CD19 (1D3), CD23 (B3B4), CD43 (S11), CD45R/B220 (RA3-6B2), CD45.1 (A20), CD45.2 (104), CD93 (AA4.1), CD138 (281-2), IgM (RMM-1), Ly6G (1A8), MHC-II (M5/114.15.2). For dead cell exclusion, LIVE/DEAD® fixable Near-IR stain kit (Invitrogen, Carlsbad, USA) was used. For intracellular staining, Foxp3 staining kit (eBioscience, Thermo Fisher, Waltham, USA) was used for Foxp3 (FKJ-16s), GM-CSF (MP1-22E9), IL-6 (MP5-20F3), IL-10 (JES5-16E3), IL-17 (TC11-18H10), IFN-γ (XMG1.2), Ki67 (16A8) and NOS2 (CXNFT). All antibodies were purchased from either Biolegend, eBioscience or BD Biosciences (Franklin Lakes, USA). For intracellular cytokine staining, cells were stimulated in culture medium containing phorbol 12-myristate 13-acetate (PMA, 20 ng/ml, Sigma-Aldrich), ionomycin (1 μg/ml, Sigma-Aldrich), and monensin (GolgiStop 1 μl/ml, BD Biosciences) at 37 °C/5% CO2 for 2 h. Flow cytometric analysis was performed on a CyAn ADP or CytoFLEX flow cytometer (Beckman Coulter, Brea, USA) or a FACS Aria III (BD Biosciences), and flow cytometric data were analyzed using FlowJo software (Tree Star, Ashland, USA). FACS sorting was performed on a FACS Aria III (BD Biosciences).
Knier B., Hiltensperger M., Sie C., Aly L., Lepennetier G., Engleitner T., Garg G., Muschaweckh A., Mitsdörffer M., Koedel U., Höchst B., Knolle P., Gunzer M., Hemmer B., Rad R., Merkler D, & Korn T. (2018). Myeloid-derived suppressor cells control B cell accumulation in the CNS during autoimmunity. Nature immunology, 19(12), 1341-1351.
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3

Comprehensive Immune Cell Phenotyping

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Cell suspensions were treated with Fc block CD16/CD32 (2.4G2) and surface markers were stained with fluorochrome conjugated anti-mouse CD3 (145-2C11), CD4 (RM4-5), CD11b (M1/70), CD19 (1D3), CD23 (B3B4), CD43 (S11), CD45R/B220 (RA3-6B2), CD45.1 (A20), CD45.2 (104), CD93 (AA4.1), CD138 (281-2), IgM (RMM-1), Ly6G (1A8), MHC-II (M5/114.15.2). For dead cell exclusion, LIVE/DEAD® fixable Near-IR stain kit (Invitrogen, Carlsbad, USA) was used. For intracellular staining, Foxp3 staining kit (eBioscience, Thermo Fisher, Waltham, USA) was used for Foxp3 (FKJ-16s), GM-CSF (MP1-22E9), IL-6 (MP5-20F3), IL-10 (JES5-16E3), IL-17 (TC11-18H10), IFN-γ (XMG1.2), Ki67 (16A8) and NOS2 (CXNFT). All antibodies were purchased from either Biolegend, eBioscience or BD Biosciences (Franklin Lakes, USA). For intracellular cytokine staining, cells were stimulated in culture medium containing phorbol 12-myristate 13-acetate (PMA, 20 ng/ml, Sigma-Aldrich), ionomycin (1 μg/ml, Sigma-Aldrich), and monensin (GolgiStop 1 μl/ml, BD Biosciences) at 37 °C/5% CO2 for 2 h. Flow cytometric analysis was performed on a CyAn ADP or CytoFLEX flow cytometer (Beckman Coulter, Brea, USA) or a FACS Aria III (BD Biosciences), and flow cytometric data were analyzed using FlowJo software (Tree Star, Ashland, USA). FACS sorting was performed on a FACS Aria III (BD Biosciences).
Knier B., Hiltensperger M., Sie C., Aly L., Lepennetier G., Engleitner T., Garg G., Muschaweckh A., Mitsdörffer M., Koedel U., Höchst B., Knolle P., Gunzer M., Hemmer B., Rad R., Merkler D, & Korn T. (2018). Myeloid-derived suppressor cells control B cell accumulation in the CNS during autoimmunity. Nature immunology, 19(12), 1341-1351.
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4

Multicolor Flow Cytometry Immunophenotyping

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Cell suspensions were labeled directly with the following fluorochrome‐conjugated anti‐mouse antibodies purchased from either BioLegend, BD Biosciences, or eBioscience: CD3ε (500A2), CD4 (RM4‐5), CD8α (53‐6.7), CD11c (N418), CD19 (6D5), CD25 (PC61.5), CD44 (IM7), CD45 (30‐F11), CD45.1 (A20), CD45.2 (104), CD49b (DX5), CD80 (16‐10A1), CD90.2 (53‐2.1), CD172α (P84), CD326 (G8.8), anti‐human CD2 (RPA‐2.10), Ly51 (6C3), F4/80 (BM8), I‐A/I‐E (M5/114.15.2), and XCR1 (ZET). UEA‐1 was labeled with a biotinylated anti‐mouse antibody (clone U1216; Vector Labs) and subsequently detected with a fluorochrome‐conjugated streptavidin. To block Fc receptors, the staining mix always contained unconjugated anti‐FcRγIII/II antibody (BioXcell; final concentration 10 µg ml−1). For exclusion of dead cells, either 4′,6‐diamidine‐2′‐phenylindole dihydrochloride (Merck) or LIVE/DEAD™ Fixable Near‐IR Stain Kit (Invitrogen) was used. Stained cells were assessed by LSRFortessa™ flow cytometer (BD Biosciences) and data was analyzed with FlowJo® Software (TreeStar).
Herppich S., Beckstette M, & Huehn J. (2021). The thymic microenvironment gradually modulates the phenotype of thymus‐homing peripheral conventional dendritic cells. Immunity, Inflammation and Disease, 10(2), 175-188.
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5

Comprehensive T Cell Immunophenotyping

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Staining was performed with human FC block, LIVE/DEAD Fixable Near-IR Stain Kit (Invitrogen), and fluorochrome-conjugated anti-human surface markers (CD3, CD4, CD8, CD14, CD15, CD19, CD33, CD39, CD45, CD45RA, CD45RO, CD96, CD161/KLRB1, CD183/CXCR3, CD185/CXCR5, CD194/CCR4, CD196/CCR6, CD197/CCR7, CD279/PD1, HLA-DR, Lox-1, and TCR γδ), all from BioLegend except for CD4, CD161 (BD Biosciences), and CD45RO (Beckman Coulter). Fluorescence minus one (FMO) staining was performed with all blood samples. Advanced cell-surface characterization of T cell subsets was performed on an Aurora spectral analyzer (Cytek). For RNA analysis, cells were sorted into TCL buffer (Qiagen) with a FACSAria III machine (BD Biosciences) and stored at −80 °C until further use.
Mitsdoerffer M., Aly L., Barz M., Engleitner T., Sie C., Delbridge C., Lepennetier G., Öllinger R., Pfaller M., Wiestler B., Rad R., Meyer B., Knier B., Schmidt-Graf F., Gempt J, & Korn T. (2022). The glioblastoma multiforme tumor site promotes the commitment of tumor-infiltrating lymphocytes to the TH17 lineage in humans. Proceedings of the National Academy of Sciences of the United States of America, 119(34), e2206208119.
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6

Multiparametric Flow Cytometry Analysis of Lymphoid Populations

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Cell suspensions from lymphoid organs were stained with fluorochrome-conjugated anti-mouse CD4 (RM4-5), CD25 (PC61.5), CD44 (IM7), CD45.1 (A20), CD45.2 (104), CD62L (MEL-14), CD90.1 (OX-7), GITR (CD357) (DTA-1), Ki67 (16A8), CD126 (D7715A7) and KLRG1 (2F1), which were purchased from either Biolegend, eBioscience or BD Biosciences. For dead cell exclusion, LIVE/DEAD® fixable Near-IR stain kit (Invitrogen) was used. For intracellular staining, Foxp3 staining kit (eBioscience) was used for Foxp3 (FKJ-16 s), IL-10 (JES5-16E3), IL-17 (TC11-18H10), IFN-γ (XMG1.2). For cytokine stainings, cells were stimulated in culture medium containing phorbol 12-myristate 13-acetate (PMA, 20 ng/ml, Sigma), ionomycin (1 μg/ml, Sigma), and monensin (GolgiStop 1 μl/ml, BD Biosciences) at 37°C/5% CO2 for 4 h. Flow cytometric analysis was performed on a CyAn ADP flow cytometer (Beckman Coulter) or a FACS Aria III (BD Biosciences), and flow cytometric data were analyzed using FlowJo software (Tree Star).
Garg G., Muschaweckh A., Moreno H., Vasanthakumar A., Floess S., Lepennetier G., Oellinger R., Zhan Y., Regen T., Hiltensperger M., Peter C., Aly L., Knier B., Palam L.R., Kapur R., Kaplan M.H., Waisman A., Rad R., Schotta G., Huehn J., Kallies A, & Korn T. (2019). Blimp1 Prevents Methylation of Foxp3 and Loss of Regulatory T Cell Identity at Sites of Inflammation. Cell Reports, 26(7), 1854-1868.e5.
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7

Proliferation and Viability Assay

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Cells were cultured in complete Keratinocyte Growth Medium II (Promocell GmbH) at 7% CO2 and 36°C. 647 EdU Click Proliferation Kit (BD, #565456) was used according to datasheet and dead cells were stained with LIVE/DEAD™ Fixable NearIR Stain Kit, for 633/635 nm excitation (Life Technologies). Data were acquired on a BD LSRII Fortessa and analyzed with Flowjo software.
Le A., Azouz A., Thomas S., Istaces N., Nguyen M, & Goriely S. (2021). JNK1 Signaling Downstream of the EGFR Pathway Contributes to Aldara®-Induced Skin Inflammation. Frontiers in Immunology, 11, 604785.
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8

Growth Kinetics and Viability Assay

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For growth rate analysis, four replicate tubes were used per treatment. On days 0, 5 and 10, a small aliquot of cell suspension was harvested from each tube, labelled with Trypan Blue 0.4 % w/v and counted using a haemocytometer. A test of cell viability was also performed on day 5 of treatment using the LIVE/DEAD Fixable Near-IR stain kit (Life Technologies). Cells were stained according to the manufacturer’s instructions and analysed by flow cytometry. Flow cytometry was performed using a BD FACSVerse (BD Biosciences, Stockholm, Sweden) equipped with 488 nm blue and 633 nm red lasers, and results were analysed using the FACSDiva (BD Biosciences) software. Cells frozen at -80 °C and thawed three times were used as negative controls.
Mangia C., Vismarra A., Kramer L., Bell-Sakyi L., Porretta D., Otranto D., Epis S, & Grandi G. (2016). Evaluation of the in vitro expression of ATP binding-cassette (ABC) proteins in an Ixodes ricinus cell line exposed to ivermectin. Parasites & Vectors, 9, 215.
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9

Skin cell isolation and sorting

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Skin samples were incubated with dispase II (Sigma-Aldrich, 2.4 mg/ml), collagenase IV (Worthington, 0.4 mg/ml) and DNase I (Sigma-Aldrich, 100 μg/ml) for 2h at 37°C. Dead cells were stained with LIVE/DEAD™ Fixable NearIR Stain Kit, for 633/635 nm excitation (Life Technologies). Then, they were incubated with rat anti-mouse CD16/CD32 (BD, 2.4G2, dilution 1:100, 553141), and a surface staining antibody mix CD45-PE (BD, 30F11, dilution 1:100, 553081), CD31-PE (BD, MEC 13.3, dilution 1:100, 561073), CD140a-PE (BD, APA5, dilution 1:100, 562776), EpCam-BV421 (BD, G8.8, dilution 1:100, 563214), and CD49f-PerCpCy5.5 (BD, GoH3, dilution1:100, 562495). Cells were sorted on a BD FACSAria™ III.
Le A., Azouz A., Thomas S., Istaces N., Nguyen M, & Goriely S. (2021). JNK1 Signaling Downstream of the EGFR Pathway Contributes to Aldara®-Induced Skin Inflammation. Frontiers in Immunology, 11, 604785.
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