Ghost red 780

Manufactured by Cytek Biosciences

Sourced in United States

The Ghost Red 780 is a laser that emits light at a wavelength of 780 nanometers. It is a core component of flow cytometry instruments and is commonly used for the detection and analysis of fluorescently labeled cells or particles.

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Ghost DyeTM Red 780 Ghost Dye Red 780 viability dye Ghost Dye Red 780 Ghost Red 780 Viability Dye Ghost 780 Ghost-Red Red 780

8 protocols using ghost red 780

Antibody Repertoire Profiling in Tumor-Bearing Mice

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Four days before infection, sera were collected from individual mice for evaluation of preinfection antibody repertoire. Ten days postinfection, a second blood sample provided sera for postinfection antibody assays. The same procedure was followed for control PBS-injected mice. Sera were diluted at 1:60 in PBS. 1 × 10⁵ LLC or EL4 tumor cells were plated in a 96-well plate and stained with a viability dye (1:1000 dilution in PBS, Ghost Red 780, #13-0865, TONBO Biosciences, San Diego CA, USA). After washing, cells were stained on ice for 1 hour with 60 µL of the diluted pre- or postinfection sera. Cells were then stained on ice for 30 minutes with FITC-conjugated goat anti-mouse IgG (1:100, Invitrogen, Carlsbad, CA, USA) as the secondary antibody. Samples were run on a Fortessa flow cytometer, and 30,000 events were recorded. Controls for nonspecific background included live/dead stained LLC and EL4 cells and secondary antibody only staining for each cell type.

Jacqueline C., Dracz M., Xue J., Binder R.J., Minden J, & Finn O. (2022). LCVM infection generates tumor antigen-specific immunity and inhibits growth of nonviral tumors. Oncoimmunology, 11(1), 2029083.

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Flow Cytometric Characterization of Tumor Cell Lines

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At the indicated timepoints, 2×10⁵ to 10⁶ MEPiC or MCF10A cells were trypsinized, collected, washed and stained with a viability dye (1:1000 dilution in PBS, Ghost Red 780, #13-0865, TONBO Biosciences, San Diego CA, USA) for 15 minutes at 4 °C. Cells were fixed with a fixation/permeabilization solution (Cytofix/Cytoperm™, Cat No: 554715, BD Biosciences, Franklin Lakes, NJ, USA) for 20 minutes at 4 °C. Cells were stained with anti-CEA antibody (1:100), anti-MUC1 antibody (1:100), trastuzumab (1:2000), and H14K6 (1:200) diluted in flow cytometry buffer (PBS + 1% BSA) for 30 minutes at 4 °C, followed by two washes with BD wash buffer. Cells were then stained with secondary antibodies (see ‘Antibodies” section; 1:200 dilution in flow cytometry buffer) for 30 minutes at 4 °C. Samples were run on a BD Fortessa flow cytometer and 30,000 total events were recorded per sample. Samples were gated based on the negative signal for APC-Cy7 (i.e., live cells) and APC (human) or FITC (mouse) mean fluorescence intensities (MFIs) were measured using FlowJo (BD).

Jacqueline C., Lee A., Frey N., Minden J.S, & Finn O.J. (2020). Inflammation-induced, abnormal expression of self-molecules on epithelial cells: targets for tumor immunoprevention. Cancer immunology research, 8(8), 1027-1038.

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Comprehensive Immunological Antibody Panel

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The following antibodies were used in this study: anti-B7-H3 (FM276, Miltenyi Biotech), anti-GD2 (14.G2a, BD Biosciences), human Ig (polyclonal, Thermo Fisher Scientific), anti-mouse IgG (polyclonal, R&D), anti-CD3 (UCHT1, BioLegend), anti-HisTag (J095G45, BioLegend), anti-CD34 (QBEnd10, R&D), anti-ab-TCR (IP26, BioLegend), anti-CD107a (H4A3, BioLegend), anti-cD25 (BC96, BioLegend), anti-CD69 (FN50, BioLegend), anti-Tim3 (F38-2E2, BioLegend), anti-Lag3 (11C3C65, BioLegend), anti-PD-1 (EH12.1, BD Biosciences), anti-mouse CD45 (30-F11, BioLegend), anti-human CD45 (HI30, BioLegend), Ghost Red 780 (Tonbo Biosciences), Zombie Yellow Viability Dye (BioLegend), propidium iodide (Gibco), Cell Trace Violet (Thermo Fisher Scientific), and Precision Count Beads (BioLegend).

Birley K., Leboreiro-Babe C., Rota E.M., Buschhaus M., Gavriil A., Vitali A., Alonso-Ferrero M., Hopwood L., Parienti L., Ferry G., Flutter B., Himoudi N., Chester K, & Anderson J. (2022). A novel anti-B7-H3 chimeric antigen receptor from a single-chain antibody library for immunotherapy of solid cancers. Molecular Therapy Oncolytics, 26, 429-443.

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Multiparametric Flow Cytometry Analysis

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Cells were trypsinized, washed, collected and then stained with a viability dye (1:1000 dilution in PBS, Ghost Red 780, #13-0865, TONBO Biosciences, San Diego CA, USA) for 15 min at 4°C. Cells were stained with the anti-MUC1 antibody 4H5 (1:100), anti-HER2 antibody trastuzumab (1:2000), anti-CEACAM1 antibody (1:100), anti-Serpin B1 (1:100), anti-Annexin A1 (1:400), anti-CRT (1:100), anti-PECAM1 (1:100) and anti-CD47 (100) diluted in flow cytometry buffer (PBS + 1% BSA), for 30 min at 4°C followed by two washes with FACS buffer. Cells were then stained with secondary goat anti-mouse IgG or F(ab’)2 anti-human IgG (1:200 dilution in FACS buffer) for 30 min at 4°C. Samples were run on a Fortessa flow cytometer and gated based on negative signal for APC-Cy7 (i.e., live cells). APC (human) or FITC (mouse) mean fluorescence intensities were measured. 30,000 total events were recorded per sample.

Jacqueline C., Dracz M., Boothman S., Minden J.S., Gottschalk R.A, & Finn O.J. (2021). Identification of Cell Surface Molecules That Determine the Macrophage Activation Threshold Associated With an Early Stage of Malignant Transformation. Frontiers in Immunology, 12, 749597.

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Macrophage-Mediated Phagocytosis Assay

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Macrophages and MCF cells were washed twice in PBS, and labeled with 1 µM CellTrace Yellow, Violet or CSFE (ThermoFisher, Waltham, MA, USA) according to manufacturer’s protocol. After 2 hours of co-incubation in 6-well plates, cells were stained with a viability dye (1:1000 dilution in PBS, Ghost Red 780, #13-0865, TONBO Biosciences, San Diego CA, USA) for 15 min at 4°C. Samples were run on a Fortessa flow cytometer and gated based on negative signal for APC-Cy7 (i.e., live cells). AMNIS image cytometry was performed using an Amnis cytometer. Cells were first visualized in a bright field and identified as macrophages (PacBlue) or MCF cells (FITC). The IDEAS software 6.2 was used to evaluate the percentage of doublets using the same gating strategy as for the phagocytosis assay. The images were merged to confirm the uptake of MCF cells by macrophages. The internalization score was measured using the internalization wizard of the IDEAS software.

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Comprehensive PBMC Phenotyping by Flow Cytometry

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Peripheral blood mononuclear cells (PBMC) were isolated over a Ficoll-Hypaque cushion (GE, Pittsburgh, PA) from EDTA-contained blood, aliquot, and stored at -80°C before use. Antibodies were incubated with PBMC at 4°C for 30 min for surface staining. After surface staining, the cells were washed and analyzed by flow cytometry. The following fluorochrome-labeled monoclonal antibodies were used (clone): anti-human CD3-percp (OKT3), anti-CD56-APC (B-159), anti-CD16-PEcy7 (3G8), anti-human CD4-BV421 (RPA-T4), anti-human CD107a-BV500 (H4A3), anti-human CD38-FITC (HIT2), anti-human HLA-DR-PE (G46-6), anti-human NKG2D-BV500 (1D11), and Ghost Red 780 (Tonbo Biosciences, San Diego, CA). Mouse IgG1-BV500, IgG1-APC and IgG2a-PE isotype antibodies were used to gate on CD107a/NKG2D-BV500, CD38 and HLA-DR respectively. Cells were collected by BD FACSVerse Flow Cytometer (BD Biosciences) and data were analyzed by FlowJo software (Version 10.0.8).

Luo Z., Li Z., Martin L., Hu Z., Wu H., Wan Z., Kilby M., Heath S.L., Huang L, & Jiang W. (2017). Increased Natural Killer Cell Activation in HIV-Infected Immunologic Non-Responders Correlates with CD4+ T Cell Recovery after Antiretroviral Therapy and Viral Suppression. PLoS ONE, 12(1), e0167640.

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Retinal Cell Isolation and Sorting

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Three days after optic nerve crush, retinas were dissociated using papain digestion (17 U/ ml papain, Worthington; 5.5 mM L-cysteine; 0.006% DNase; 1.1 mM EDTA in DMEM/2% B27) during 30 min at 37 °C as previously described at [35 (link)]. After digestion, retinas were washed in DMEM, gently triturated using a Pasteur pipet and centrifuged at 300 g × 5 min at RT. Dissociated cells were resuspended in DMEM/2% B27, passed through a 35 μm cell strainer and placed on ice until use. Dissociated retinal cells were stained with Ghost red 780 (TONBO Biosciences) to exclude non-viable cells and then CFP cells were separated by BD FACS SORP Aria-IIu (BD Biosciences) at Flow Cytometry Shared Resource, University of Miami, Sylvester Comprehensive Cancer Center. C57BL6/J retinal cells were used as unstained control. Sorted cells were collected in PBS and immediately frozen at − 80 °C.

Ayupe A.C., Beckedorff F., Levay K., Yon B., Salgueiro Y., Shiekhattar R, & Park K.K. (2021). Identification of long noncoding RNAs in injury-resilient and injury-susceptible mouse retinal ganglion cells. BMC Genomics, 22, 741.

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Multiparameter Flow Cytometry Analysis

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All flow cytometry analysis was conducted in the University of Minnesota Flow Cytometry Core Facility using BD LSR II and Fortessa cytometers (BD Biosciences). For surface staining, cells were stained for 20 min with fluorochrome-conjugated antibodies before washing and analysis or intracellular staining. Intracellular detection of FOXP3 was performed using the eBioscience Transcription Factor staining kit, with the fixation/perm incubation being performed at room temperature for 30 minutes and the intracellular staining being performed in 1xPermeablization buffer for 30 minutes at room temperature. Antibodies to mouse- CD4 (BV786- GK1.5- Biolegend, BV650- L3T4- Biolegend- 1:200), CD8 (BV786– 53-6.7- BD Biosciences, Biotin- 53–6.7- Tonbo Biosciences- 1:200), Ter119 (BD Biosciences, 1:200), CD25 (PerCP-Cy5.5- PC61.5- Tonbo Biosciences, BV421-PC61.5- Biolegend- 1:200), FOXP3 (AF488- FJK-16s- eBiosciences- 1:100), CD73 (PE-Cy7- TY/11.8- Biolegend, eF450- TY/11.8-eBiosciences- 1:200), TCR Vα2 (APC- B20.1- eBiosciences), TIM3 (PE- RMT3–23- Biolegend, 1:100), TIGIT (PE- GIGD7- eBiosciences, 1:100), ST-2 (PerCP-eF710- RMST2–33- eBioscience, 1:100), LAG3 (PE- C9B7W- eBiosciences, 1:100) and Ghost Red 780 (Tonbo Biosciences, 1:1000) were used for flow cytometry analysis.

Owen D.L., La Rue R.S., Munro S.A, & Farrar M.A. (2022). Tracking regulatory T cell development in the thymus using scRNA-Seq/TCR-Seq. Journal of immunology (Baltimore, Md. : 1950), 209(7), 1300-1313.

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