Ghost dye red 780

Manufactured by Cytek Biosciences

Sourced in United States

Ghost Dye Red 780 is a fluorescent dye used in flow cytometry applications. It is designed to stain dead or dying cells, allowing for the identification and exclusion of these cells from analysis.

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Lab products found in correlation

Attune nxt flow cytometer, by Thermo Fisher Scientific (10 mentions) Lsrfortessa, by BD (8 mentions) Foxp3 transcription factor staining buffer set, by Thermo Fisher Scientific (5 mentions) Lsr 2, by BD (5 mentions) 96 well autosampler, by Thermo Fisher Scientific (5 mentions) Cytofix cytoperm kit, by BD (4 mentions) Pharmingen transcription factor phospho buffer set, by BD (4 mentions) Dapi solution, by Thermo Fisher Scientific (3 mentions) Accucheck counting beads, by Thermo Fisher Scientific (3 mentions) T bet 4b10, by BioLegend (3 mentions) Dnase 1, by Merck Group (3 mentions) Phorbol myristate acetate (pma), by Merck Group (3 mentions) Facsaria, by BD (3 mentions) Foxp3 transcription factor staining buffer kit, by Cytek Biosciences (3 mentions) Fortessa, by BD (3 mentions) Anti ki67 fitc, by BD (2 mentions) Mouse serum, by Merck Group (2 mentions) Pe anti human cd279 pd 1 eh12.2h7, by BioLegend (2 mentions) Cd34 apc, by BioLegend (2 mentions) Methocult optimum, by STEMCELL (2 mentions)

Spelling variants of this product

Ghost Dye (26 citations) Ghost Dye 780 (14 citations) Ghost Red 780 (8 citations) Ghost 780 (6 citations) Ghost-Red (6 citations) Red 780 (2 citations) Ghost DyeTM Red 780 (2 citations)

89 protocols using ghost dye red 780

Multicolor Flow Cytometry for Cell Characterization

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Fluorescence minus one (FMO) tubes, rainbow bead tubes, and single antibody tubes were prepared as gating references. Antibodies were pipetted into flow cytometry tubes according to manufacturer’s instructions. Antibodies used include CD34 = Brilliant Violet 421 (BioLegend, San Diego, CA, United States), CD45 = Alexa Fluor 488 (BioLegend, San Diego, CA, United States), CD133 = PE (Miltenyi Biotec, North Rhine-Westphalia, Germany) CD31 = Brilliant Violet 605 (BioLegend, San Diego, CA, United States), CD105 = PE-Cy7 (BioLegend, San Diego, CA, United States), Ghost Dye Red 780 = Tonbo Biosciences, San Diego, CA.
Flow cytometry was performed by blinded scientists on a ThermoFisher Attune NxT (Waltham, MA, United States). Samples were analyzed by blinded scientists using FlowJo software (FlowJo, Ashland, OR, United States).

MacLaughlin K.J., Barton G.P., Braun R.K., MacLaughlin J.E., Lamers J.J., Marcou M.D, & Eldridge M.W. (2023). Hyperbaric air mobilizes stem cells in humans; a new perspective on the hormetic dose curve. Frontiers in Neurology, 14, 1192793.

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In Vitro Immune Cell Assay

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Seven days after immunization, single cell suspensions generated from spleens were subjected to ACK red blood cell lysis and counted using a Vi-Cell automated cell counter (Beckman Coulter). For in vitro stimulation assays, 1 × 10⁶ cells were incubated with 1 μg/ml peptide and 3 μg/ml brefeldin A for 5 h at 37°C in complete media (RPMI 1640 containing 10% FBS, 10 mM HEPES, 0.1 mM β-ME, 0.1 mM non-essential amino acids, 0.1 mM sodium pyruvate, 2 mM L-glutamine and penicillin-streptomycin). After stimulation, cells were surfaced-stained with CD8α-BV421 (clone 53.67, BioLegend), CD4-FITC (GK1.5, BioLegend), B220-PE-Cy7 (clone RA3-6B2, Tonbo), and a fixable viability dye (Ghost Dye Red 780, Tonbo) for 10 min at room temperature. After staining for surface antigens, cells were fixed and permeabilized with FoxP3 fixation/permeabilization buffers (Tonbo) for 15 min at room temperature. After fixation and permeabilization, cells were washed in perm/wash buffer and stained for intracellular cytokines using IFNγ-APC (XMG1.2, Tonbo) and TNFα-PE (MP6-XT22, BD Biosciences) diluted in perm/wash buffer for 30 min at room temperature. After a final wash, flow cytometry data were acquired on a four-laser (405, 488, 561, 638 nm) CytoFLEX S flow cytometer (Beckman Coulter) and analysis was performed using FlowJo (version 10.7.1; BD Biosciences).

Davenport B.J., Morrison T.E., Kedl R.M, & Klarquist J. (2021). Conserved and novel mouse CD8 T cell epitopes within SARS-CoV-2 spike RBD protein identified following subunit vaccination. Journal of immunology (Baltimore, Md. : 1950), 206(11), 2503-2507.

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Multicolor Flow Cytometric Analysis of Immune Cells

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Single-cell suspension of different lymphoid organs as well other tissues including lung and lamina propria were prepared as described previously (6 (link)). For FACS analysis, cells were stained with Ghost Dye Red 780 (Tonbo Biosciences) or Fixable Viability Dye eflour 450 (eBiosciences) followed by surface staining with antibodies against CD4, CD8, CD62L, CD44, Ly5.1. Intracellular staining was performed with antibodies against Foxp3, T-bet, GATA3, RORγT (eBiosciences), TCF1 (Cell signaling) and GFP (eBioscience). To assess IFNγ, IL-17, IL-4 production, cells were incubated with PMA (50ng/ml), ionomycine (0.5μg/ml) and Brefeldin A (1μg/ml) for 4 h at 37 °C, followed by standard staining as described above. BD LSRFortessa or BD LSRFortessa 20x cell analyzer (BD Biosciences) was used for data collection while Flowjo software (Tree Star) was used for data analysis.

Cho S., Wu C.J., Nguyen D.T., Lin L.L., Chen M.C., Khan A.A., Yang B.H., Fu W, & Lu L.F. (2017). A novel miR-24-TCF1 axis in modulating effector T cell responses. Journal of immunology (Baltimore, Md. : 1950), 198(10), 3919-3926.

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Evaluating Cell Proliferation by Flow Cytometry

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0.5 × 10⁶ CLBL-1 cells were harvested after 72-hour of DMSO or pevonedistat treatment. Cells first were stained with the fixable viability dye, Ghost Dye Red 780 (1:1000; Tonbo Biosciences, San Diego, CA). For intracellular Ki67 staining, cells were fixed and permeabilized using the BD Cytofix/Cytoperm kit (BD Biosciences, San Jose, CA) and further labelled with FITC anti-Ki67 (1:20; BD Biosciences, San Jose, CA). DAPI solution (1:250; Thermo Fisher Scientific, Madison, WI) was then added and incubated overnight at 4°C. Samples were analysed on a BD LSRFortessa (BD Biosciences, San Jose, CA), and results were analysed using FlowJo v10.0.7 software.

Assumpção A.L., Lu Z., Marlowe K.W., Shaffer K.S, & Pan X. (2018). Targeting NEDD8-activating enzyme is a new approach to treat canine diffuse large B-cell lymphoma. Veterinary and comparative oncology, 16(4), 606-615.

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Flow Cytometry Analysis of PD-1 Expression

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The protein levels were measured after different time points of incubation by flow cytometry in a BD FACS Canto II Flow Cytometer and analysed with FlowJo software (FlowJo, LLC). Cells were rinsed twice with PBS (1X) and resuspended with PBS and Ghost Dye Red 780 (1:1000 dilution, Tonbo Biosciences) for 30 min on ice. After that, cells were rinsed once with PBS-0.2% Bovine Serum Albumin (BSA) (PanReac AppliChem) and resuspended in 50 μl of this buffer with the PE anti-human CD279 (PD-1) (EH12.2H7, 1:100 dilution, BioLegend) antibody for 30 min on ice. The analysis strategy was to compare the mean fluorescence intensity between treated and untreated live cells for oxLDL experiments. For anti-CD69 monoclonal antibody engagement experiments, samples were incubated with mouse serum (1:100 dilution, Sigma-Aldrich) for 15 min before adding the same volume with flow cytometry antibodies. Percentage of positive cells for PD-1 were represented at different times.

Jiménez-Fernández M., Rodríguez-Sinovas C., Cañes L., Ballester-Servera C., Vara A., Requena S., de la Fuente H., Martínez-González J, & Sánchez-Madrid F. (2022). CD69-oxLDL ligand engagement induces Programmed Cell Death 1 (PD-1) expression in human CD4 + T lymphocytes. Cellular and Molecular Life Sciences, 79(8), 468.

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JAK Inhibitor Effects on CLBL-1 Cell Proliferation

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After 72‐hour JAK inhibitor treatment, 0.5 × 10⁶ CLBL‐1 cells were harvested. Cells first were stained with the fixable viability dye, Ghost Dye Red 780 (Tonbo Biosciences20; 1 : 1000). For intracellular Ki67 staining, cells were fixed and permeabilized with the BD Cytofix/Cytoperm kit (BD Biosciences17) and further labeled with FITC anti‐Ki67 (BD Biosciences17; 1 : 20). DAPI solution (Thermo Fisher Scientific10; 1 : 250) then was added and incubated overnight at 4°C. Samples were analyzed the next day on a BD LSRFortessa (BD Biosciences17) and results were analyzed with FlowJo v10.0.7 software18.

Lu Z., Hong C.C., Jark P.C., Assumpção A.L., Bollig N., Kong G, & Pan X. (2017). JAK1/2 Inhibitors AZD1480 and CYT387 Inhibit Canine B‐Cell Lymphoma Growth by Increasing Apoptosis and Disrupting Cell Proliferation. Journal of Veterinary Internal Medicine, 31(6), 1804-1815.

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MSCV-based Retroviral Vector Targeting CD4

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Two MSCV-based γ retroviral vectors were used to target the Cd4 gene as described by Kotov et al. ²⁹. One vector encoded Cas9 and the fluorescent protein mNeongreen and the other either Cd4 or control LacZ guide RNAs (gRNAs) and the fluorescent protein mAmetrine ³⁰. Both vectors were produced by modifying the LMP-Amt vector from S. Crotty (La Jolla Institute) as described by Kotov et al. ²⁹. Platinum-E cells (Cell Biolabs) were grown in complete DMEM (Life Technologies) and transfected with the aforementioned plasmids. Virus-containing supernatants were harvested several days later. B3K508 T cells were cultured with complete IMDM (MilliporeSigma) containing IL-7 (Tonbo Biosciences) and then activated in plates coated with CD3 (2C11; Bio X Cell) and CD28 (37.51; Bio X Cell) antibodies. The cells were transduced with retroviral supernatant and polybrene (MilliporeSigma) 24 and 40 hours after activation and transferred to uncoated plates without CD3 or CD28 Abs for five days, the last three in complete IMDM-containing IL-7 (Tonbo Biosciences). The cells were then stained with P5R:I-A^b or P5R:I-A^b-4E tetramers and then BV786-labeled CD4 (RM4–5; BD Biosciences) antibody and a fixable viability dye (Ghost Dye Red 780; Tonbo Biosciences). The stained cells were analyzed by flow cytometry as described below.

Dileepan T., Malhotra D., Kotov D.I., Kolawole E.M., Krueger P.D., Evavold B.D, & Jenkins M.K. (2021). MHC class II tetramers engineered for enhanced binding to CD4 improve detection of antigen-specific T cells. Nature biotechnology, 39(8), 943-948.

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CD34+ HSPC Colony Assay

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Two days post targeting, HSPCs were stained using CD34 APC (no. 561, BioLegend) and Ghost Dye Red 780 (Tonbo Biosciences), and live CD34⁺ cells were sorted into 96-well plates containing MethoCult Optimum (STEMCELL Technologies). After 12–16 d, colonies were appropriately scored based on external appearance in a blinded fashion.

Cromer M.K., Camarena J., Martin R.M., Lesch B.J., Vakulskas C.A., Bode N.M., Kurgan G., Collingwood M.A., Rettig G.R., Behlke M.A., Lemgart V.T., Zhang Y., Goyal A., Zhao F., Ponce E., Srifa W., Bak R.O., Uchida N., Majeti R., Sheehan V.A., Tisdale J.F., Dever D.P, & Porteus M.H. (2021). Gene replacement of α-globin with β-globin restores hemoglobin balance in β-thalassemia-derived hematopoietic stem and progenitor cells. Nature medicine, 27(4), 677-687.

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Tetramer-Based Immune Cell Profiling

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Spleen and lymph node cells or blood cell were harvested and stained with p:I-A^b tetramers, and in a subset of experiments with CXCR5 (L138D7; BioLegend) antibody for one hour at room temperature, then incubated with anti-PE and anti-APC magnetic beads. Samples then passed over magnetized columns (Miltenyi). Bound cells were eluted from the columns and stained for 30 minutes on ice with antibodies from eBioscience to CD3e (145-2C11), CD4 (GK1.5; BD), CD8 (53-6.7), CD90.1 (HIS51;), CD90.2 (53-2.1), CD11b (M1/70), CD11c (N418), F4/80 (BM8), CD44 (IM7), CD45.2 (104), B220 (RA3-6B2), and CD45.1 (A20). Cellular viability by GhostDye Red 780 (Tonbo). Stained cells were fixed and permeabilized with the Foxp3/Transcription Factor Staining Buffer Set (eBioscience) for one hour at room temperature and stained overnight at 4 °C with antibodies to T-bet (4B10; BioLegend), FoxP3 (FJK-16s; eBioscience), RORγt (Q31-378; BD), and/ or Bcl-6 (K112-91; BD). Cells were analyzed on a Fortessa X-20 (BD) using FlowJo software v10 (TreeStar). The total number of tetramer-positive cells in the enriched fraction from each mouse was determined using AccuCheck Counting Beads (ThermoFisher).

Burrack A.L., Malhotra D., Dileepan T., Osum K.C., Swanson L.A., Fife B.T, & Jenkins M.K. (2017). Allograft rejection is associated with weak T cell responses to many different graft leukocyte-derived peptides. Journal of immunology (Baltimore, Md. : 1950), 200(2), 477-482.

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Flow Cytometric Immune Profiling

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Murine cells were incubated with a Ghost Dye Red 780 (Tonbo Biosciences) viability marker and anti-mouse CD16/32 (Tonbo Biosciences) in FACS buffer for 30 minutes on ice to block murine Fc receptors. Cells were then stained with the following fluorophore-conjugated anti-mouse monoclonal antibodies: CD45, CD3, CD4, CD8, ICOS [C398.4A], CD11c, CD11b, GR1 [RB6-8C5], F4-80, CD206 [C068C2], B7-1, MHC-I [34-1-2S & 28-8-6], MHC-II [M5/114.15.2], PD-1, Tim3, PD-L1, and B7x [HMH4-5G1] (all from Biolegend); and SPSYVYHQF/H-2L^d Alexa-647 conjugated tetramer [NIH Tetramer Core Facility]. All antibodies were stained for an additional 45 minutes on ice. Human cells were incubated with the Ghost Dye Red 780 viability marker for 30 minutes on ice and immediately stained with the following fluorophore-conjugated anti-human monoclonal antibodies: MHC-I, MHC-II, PD-L1, and B7x [MIH43] (all from Biolegend) on ice for 45 minutes.

Ohaegbulam K.C., Liu W., Jeon H., Almo S.C, & Zang X. (2017). Tumor-expressed immune checkpoint B7x promotes cancer progression and antigen-specific CD8 T cell exhaustion and suppressive innate immune cells. Oncotarget, 8(47), 82740-82753.

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