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IR-dye is a spectrally pure near-infrared fluorescent dye designed for use in a variety of applications, including in-vivo imaging, protein labeling, and fluorescence-based assays. The dye exhibits excitation and emission maxima in the near-infrared region of the spectrum, allowing for reduced background interference and improved tissue penetration compared to visible wavelength dyes.

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Lab products found in correlation

Human ifn γ, by Thermo Fisher Scientific (2 mentions) Hanks balanced salt solution (hbss), by Thermo Fisher Scientific (2 mentions) Human ifnα, by Thermo Fisher Scientific (2 mentions) Accucountblank 15.2 mm beads, by Spherotech (2 mentions) αrabbit igg bv421 antibody, by BD (2 mentions) αcd69 pecy7, by BioLegend (2 mentions) D6w5b, by Cell Signaling Technology (2 mentions) Bovine serum albumin (bsa), by Thermo Fisher Scientific (2 mentions) Anti human cd8 clone rpa t8, by BD (2 mentions) Anti human hla a 02 clone bb7.2, by BD (2 mentions) Anti mouse tcrb constant domain clone h57 597, by BD (2 mentions) Mouse monoclonal anti flag, by Merck Group (1 mentions) Cd4 apc, by BD (1 mentions) Cd4 fitc, by BD (1 mentions) Cd19 fitc, by BD (1 mentions) Cd4 bv421, by BioLegend (1 mentions) Cd16 apc h7, by BD (1 mentions) Cd8 bv421, by BD (1 mentions) Cd107 pe, by BD (1 mentions) Cd14 apc h7, by BD (1 mentions)

Spelling variants of this product

Live/Dead Near IR dye (21 citations) Near-IR fluorescent reactive dye (9 citations) LIVE/DEAD-Near IR viability dye (6 citations) IR-dye-conjugated secondary antibodies (5 citations) LIVE/DEAD Fixable Near-IR Dead Cell Dye (5 citations) Live–Dead Fixable Dead Cell Stain near-IR-fluorescent reactive dye (4 citations) Near-IR fixable dye (4 citations) LIVE/DEAD Fixable Near-IR Dead Cell Dye staining (2 citations) LIVE/DEAD™ Fixable Near-IR Dead Cell stain dye (2 citations) LIVE/DEAD Near-IR fluorescent reactive dye (2 citations)

9 protocols using ir dye

1

Dissecting IFNγ Mediated Tumor Cell Death

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Ag- CFP+ IGS reporter tumor cells proficient or deficient for the IFNγR, were plated at 20,000 cells/well in 48-well plates and either received the indicated concentrations of human IFNα (Thermo Fisher Scientific) or human IFNγ (Invitrogen). At the indicated time points, cells were harvested, stained with IR-Dye (Invitrogen) and analyzed by flow cytometry. To measure T cell-mediated death of bystander tumor cells, a mixture of GFP+ Ag+, CFP+ Ag- IFNγR proficient and CFP+ Ag- IFNγR deficient OVCAR5 cells was plated at 100,000 cells/well in 6 well plates at a 2:1:1 ratio, and cells were then incubated with either HBSS (Gibco) or CDK4R>L-specific CD8+ T cells in HBSS at a 5:1 T cell: (total) tumor cell ratio. Cell death and total cell counts were analysed at day 3 after treatment by IR-Dye (Invitrogen) staining, and subsequent flow cytometry using AccuCountBlank 15.2 mm beads (Spherotech).
Hoekstra M.E., Bornes L., Dijkgraaf F.E., Philips D., Pardieck I.N., Toebes M., Thommen D.S., van Rheenen J, & Schumacher T.N. (2020). Long-distance modulation of bystander tumor cells by CD8+ T cell-secreted IFNγ. Nature cancer, 1(3), 291-301.
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2

Dissecting IFNγ Mediated Tumor Cell Death

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Ag- CFP+ IGS reporter tumor cells proficient or deficient for the IFNγR, were plated at 20,000 cells/well in 48-well plates and either received the indicated concentrations of human IFNα (Thermo Fisher Scientific) or human IFNγ (Invitrogen). At the indicated time points, cells were harvested, stained with IR-Dye (Invitrogen) and analyzed by flow cytometry. To measure T cell-mediated death of bystander tumor cells, a mixture of GFP+ Ag+, CFP+ Ag- IFNγR proficient and CFP+ Ag- IFNγR deficient OVCAR5 cells was plated at 100,000 cells/well in 6 well plates at a 2:1:1 ratio, and cells were then incubated with either HBSS (Gibco) or CDK4R>L-specific CD8+ T cells in HBSS at a 5:1 T cell: (total) tumor cell ratio. Cell death and total cell counts were analysed at day 3 after treatment by IR-Dye (Invitrogen) staining, and subsequent flow cytometry using AccuCountBlank 15.2 mm beads (Spherotech).
Hoekstra M.E., Bornes L., Dijkgraaf F.E., Philips D., Pardieck I.N., Toebes M., Thommen D.S., van Rheenen J, & Schumacher T.N. (2020). Long-distance modulation of bystander tumor cells by CD8+ T cell-secreted IFNγ. Nature cancer, 1(3), 291-301.
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3

Western Blot Quantification Protocol

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Following separation by SDS–PAGE, proteins transferred to nitrocellulose were detected using primary rabbit or mouse antibodies and visualized with appropriate secondary antibodies conjugated to IRDye (Thermo Fisher Scientific). Quantification was performed with ImageJ software (National Institutes of Health [NIH]).
The following antibodies were used in this study: mouse monoclonal anti-FLAG (Sigma; 1:1000); mouse monoclonal anti-Mfn2 (Sigma clone 4H8; 1:1000); mouse monoclonal anti-tubulin (Thermo Fisher Scientific clone DM1A; 1:5000); rabbit polyclonal anti-Mfn1 (gift from Jodi Nunnari, University of California, Davis; 1:500); and VDAC (Thermo Fisher Scientific, polyclonal PA1-954A; 1:1000). Briefly, Mfn1 antiserum was raised against His6-tagged fusion proteins comprised of full-length mouse dihydrofolate reductase and an internal region of Mfn1 (residues 350–580). Fusion proteins were purified on nickel nitrilotriacetic acid columns (Thermo Fisher Scientific) in 8 M urea and eluted with 0.1% SDS and 10 mM Tris-Cl, pH 7.4.
Sloat S.R., Whitley B.N., Engelhart E.A, & Hoppins S. (2019). Identification of a mitofusin specificity region that confers unique activities to Mfn1 and Mfn2. Molecular Biology of the Cell, 30(17), 2309-2319.
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4

Comprehensive Immune Cell Profiling

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Where indicated, PBMCs, BA/F3 cells, CD8+ T cells, dissociated tumor tissue, and T cell-tumor cell suspensions were stained in cold PBS with 0.5% BSA and EDTA (2 mM) for 20-30 minutes at 4°C with the following: live-dead fixable near-IR dead cell stain (IR-dye, Thermo Fisher Scientific), αCD3-BV711 antibody, and αFLAG-AF647 antibody (D6W5B, Cell Signaling Technology, 1:50), polyclonal αFLAG-AF647 (Cell Signaling Technology, 1:200), αFLAG-BV421 (L5, Biolegend, 1:50), or primary unlabeled αFLAG antibody (D6W5B, Cell Signaling Technology, 1:800) followed by secondary αRabbit-IgG-BV421 antibody (BD Biosciences, 1:200). In tumor – T cell coculture experiments, cells were also stained with αCD69-PeCy7 (H57-597, Biolegend, 1:100). Following staining, cells were washed three times and resuspended in cold PBS with 0.5% BSA and EDTA (2 mM) for flow cytometry.
van der Leun A.M., Hoekstra M.E., Reinalda L., Scheele C.L., Toebes M., van de Graaff M.J., Chen L.Y., Li H., Bercovich A., Lubling Y., David E., Thommen D.S., Tanay A., van Rheenen J., Amit I., van Kasteren S.I, & Schumacher T.N. (2021). Single Cell Analysis of Regions of Interest (SCARI) using a photo-sensitive tag. Nature chemical biology, 17(11), 1139-1147.
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5

Multiparametric Flow Cytometry Panel

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The following antibodies were used for flow cytometry: CD3-PerCP-Cy5.5 (clone SK7; eBioscience; used 1:20); CD4-FITC (clone RPA-T4; BD Biosciences; used 1:20), CD4-APC (clone RPA-T4; BD Biosciences; used 1:30), CD4-BV421 (clone SK3, Biolegend; used 1:100), CD8-BV421 (clone RPA-T8; BD Biosciences; used 1:50), CD14-APC-H7 (clone MoP9, BD Biosciences; used 1:100), CD16-APC-H7 (clone 3G8, BD Biosciences; used 1:100), CD19-FITC (clone 4G7, BD Biosciences; used 1:30), CD137-BV421 (clone 4B4-1; Biolegend; used 1:200), CD137-APC (clone 4B4-1; BD Biosciences; used 1:30), OX40-PE-Cy7 (clone Ber-ACT35, Biolegend), CD107-PE (clone H4A3, BD Biosciences; used 1:150) and PE-conjugated anti-mouse TCRβ constant domain (clone H57-597; BD Biosciences; used 1:150). The viability stain IR-Dye (Thermo Fisher, used 1:2,000) was used to identify live cells.
Cattaneo C.M., Battaglia T., Urbanus J., Moravec Z., Voogd R., de Groot R., Hartemink K.J., Haanen J.B., Voest E.E., Schumacher T.N, & Scheper W. (2023). Identification of patient-specific CD4+ and CD8+ T cell neoantigens through HLA-unbiased genetic screens. Nature Biotechnology, 41(6), 783-787.
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6

Quantifying Blood-borne T Cells in Skin Vaccinations

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To determine the fraction of blood-borne T cells in skin preparations of the vaccination site obtained during the effector phase, splenocytes of GFP-OT-I transgenic mice were first negatively enriched with the Mouse CD8 T Lymphocyte Enrichment Set (BD Biosciences). Subsequently, C57BL/6J-Ly5.1 animals received ∼700 naive GFP-OT-I splenocytes i.v., followed by primary DNA vaccination on Veet-depilated hind legs as described above. On day 10 after vaccination, mice received a one-time injection of 1.5 × 106 CD8+ negatively enriched mTmG-OT-I splenocytes as a reference for blood-borne T cells, 5 min before sacrificing the animals. Subsequently, blood and skin tissue was harvested, and cells were isolated from the two compartments, as described above. Single-cell suspensions were then stained with IR-dye (Thermo Fisher Scientific) and analyzed on an LSR II SORP (BD Biosciences).
Kok L., Dijkgraaf F.E., Urbanus J., Bresser K., Vredevoogd D.W., Cardoso R.F., Perié L., Beltman J.B, & Schumacher T.N. (2020). A committed tissue-resident memory T cell precursor within the circulating CD8+ effector T cell pool. The Journal of Experimental Medicine, 217(10), e20191711.
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7

Comprehensive Immune Cell Profiling

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Where indicated, PBMCs, BA/F3 cells, CD8+ T cells, dissociated tumor tissue, and T cell-tumor cell suspensions were stained in cold PBS with 0.5% BSA and EDTA (2 mM) for 20-30 minutes at 4°C with the following: live-dead fixable near-IR dead cell stain (IR-dye, Thermo Fisher Scientific), αCD3-BV711 antibody, and αFLAG-AF647 antibody (D6W5B, Cell Signaling Technology, 1:50), polyclonal αFLAG-AF647 (Cell Signaling Technology, 1:200), αFLAG-BV421 (L5, Biolegend, 1:50), or primary unlabeled αFLAG antibody (D6W5B, Cell Signaling Technology, 1:800) followed by secondary αRabbit-IgG-BV421 antibody (BD Biosciences, 1:200). In tumor – T cell coculture experiments, cells were also stained with αCD69-PeCy7 (H57-597, Biolegend, 1:100). Following staining, cells were washed three times and resuspended in cold PBS with 0.5% BSA and EDTA (2 mM) for flow cytometry.
van der Leun A.M., Hoekstra M.E., Reinalda L., Scheele C.L., Toebes M., van de Graaff M.J., Chen L.Y., Li H., Bercovich A., Lubling Y., David E., Thommen D.S., Tanay A., van Rheenen J., Amit I., van Kasteren S.I, & Schumacher T.N. (2021). Single Cell Analysis of Regions of Interest (SCARI) using a photo-sensitive tag. Nature chemical biology, 17(11), 1139-1147.
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8

Flow Cytometry Analysis of Immune Markers

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The following antibodies were used for flow cytometry: anti-human CD8, (clone RPA-T8; BD biosciences), anti-human IFNγR (CD119, Clone GIR-208; eBioscience), anti-human HLA-A*02 (clone BB7.2; BD Biosciences), anti-mouse TCRb constant domain (clone H57-597; BD Biosciences) and anti-human PD-L1 (CD274, clone MIH1, eBioscience). Cell surface expression of indicated markers was assessed by staining of cells with fluorochrome-labeled antibodies in FACS buffer (0.5% w/v bovine serum albumin (Fisher Scientific) in PBS) for 20–30 min at 4 °C, while protected from light. After incubation, cells were washed twice with FACS buffer before resuspension in FACS buffer for analysis. IR-Dye (Invitrogen) was used to allow for live cell selection.
Hoekstra M.E., Bornes L., Dijkgraaf F.E., Philips D., Pardieck I.N., Toebes M., Thommen D.S., van Rheenen J, & Schumacher T.N. (2020). Long-distance modulation of bystander tumor cells by CD8+ T cell-secreted IFNγ. Nature cancer, 1(3), 291-301.
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9

Flow Cytometry Analysis of Immune Markers

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The following antibodies were used for flow cytometry: anti-human CD8, (clone RPA-T8; BD biosciences), anti-human IFNγR (CD119, Clone GIR-208; eBioscience), anti-human HLA-A*02 (clone BB7.2; BD Biosciences), anti-mouse TCRb constant domain (clone H57-597; BD Biosciences) and anti-human PD-L1 (CD274, clone MIH1, eBioscience). Cell surface expression of indicated markers was assessed by staining of cells with fluorochrome-labeled antibodies in FACS buffer (0.5% w/v bovine serum albumin (Fisher Scientific) in PBS) for 20–30 min at 4 °C, while protected from light. After incubation, cells were washed twice with FACS buffer before resuspension in FACS buffer for analysis. IR-Dye (Invitrogen) was used to allow for live cell selection.
Hoekstra M.E., Bornes L., Dijkgraaf F.E., Philips D., Pardieck I.N., Toebes M., Thommen D.S., van Rheenen J, & Schumacher T.N. (2020). Long-distance modulation of bystander tumor cells by CD8+ T cell-secreted IFNγ. Nature cancer, 1(3), 291-301.
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