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Ghost dye 780

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Ghost Dye 780 is a fluorescent dye used in flow cytometry applications. It is designed to label and detect dead cells in a sample.

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Lab products found in correlation

Lsrii or lsr fortessa flow cytometers, by BD (5 mentions) Klrg1 2f1, by BD (2 mentions) Cytofix cytoperm kit, by BD (2 mentions) Cd44 clone im7, by BioLegend (2 mentions) Steptavidin bv650, by BioLegend (2 mentions) Cd8β clone ly 3, by BioLegend (2 mentions) Cd45.1 clone a20, by BioLegend (2 mentions) Thy1.1 clone ox 7, by BioLegend (2 mentions) Collagenase 4, by Merck Group (2 mentions) Gentlemacs dissociator, by Miltenyi Biotec (2 mentions) Cytoflex, by Beckman Coulter (1 mentions) Facsverse, by BD (1 mentions) Bovine serum albumin (bsa), by Merck Group (1 mentions) Gallios flow cytometer, by Beckman Coulter (1 mentions) Kaluza analysis 2, by Beckman Coulter (1 mentions) Anti f4 80 bm8, by Cytek Biosciences (1 mentions) Anti cd3 17a2, by Thermo Fisher Scientific (1 mentions) Anti cd69 h1.2f3, by Thermo Fisher Scientific (1 mentions) Anti cd45 30 f11, by BD (1 mentions) Anti pd l1 mih5, by Thermo Fisher Scientific (1 mentions)

Spelling variants of this product

Ghost Dye Red 780 (89 citations) Ghost Dye (26 citations) Ghost Red 780 Viability Dye (12 citations) Ghost Dye Red 780 viability dye (10 citations) Live/Dead Ghost dye 780 (7 citations) Ghost 780 (6 citations) Ghost DyeTM Red 780 (2 citations)

14 protocols using ghost dye 780

1

Quantifying HIV-1 Latent Reactivation

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The reactivation of HIV-1 from latently infected cells was determined by changes in intracellular green fluorescent protein (GFP) expression. J-Lat 10.6 cells were placed in 48-well plates with 2 × 105 cells/well and incubated with different drug concentrations for 24 h. The data obtained by a representative gating strategy are shown in Fig. S1. The determination of CD69 activation and cytotoxicity marker staining were performed as described previously [31 (link),56 (link)]. Cells were incubated with Ghost dye 780 (TONBO Biosciences, San Diego, CA, USA) for 30 min 4 °C. The cells were then stained with PE anti-human CD69 (FN50) mAb (BioLegend, San Diego, CA, USA) for 30 min on ice. Then, cells were fixed with 1% paraformaldehyde/PBS for 20 min RT and analyzed using BD FACSVerse (BD Biosciences, San Jose, CA, USA) and CytoFLEX (Beckman Coulter, Brea, CA, USA). The collected data were analyzed by FlowJo software (Tree Star, San Carlos, CA, USA).
Ishii T., Kobayakawa T., Matsuda K., Tsuji K., Ohashi N., Nakahata S., Noborio A., Yoshimura K., Mitsuya H., Maeda K, & Tamamura H. (2023). Synthesis and evaluation of DAG-lactone derivatives with HIV-1 latency reversing activity. European journal of medicinal chemistry, 256, 115449.
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2

Murine Splenocyte Phenotyping by Flow Cytometry

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Splenocytes were counted and resuspended in 1XPBS containing the cell viability indicator GhostDye 780 (Cat #: 13-0865; Tonbo Biosciences, San Diego, CA, USA) and incubated for 30 min at 4 °C. Cells were then washed twice with flow staining buffer containing 1% bovine serum albumin (BSA; Sigma-Aldrich, St. Louis, MO, USA), spun down and resuspended in flow staining buffer to a final concentration of 1 × 106 cells/mL. Next, cells were stained with fluorescently conjugated anti-mouse antibodies against CD3 (Cat #: 11-0032-82, ThermoFisher, Waltham, MA, USA), CD4 (Cat #: 56004282, Fisher Scientific, Lenexa, KS, USA), and CD8 (Cat #: 12008183, eBioscience, San Diego, CA, USA). Samples requiring intracellular staining were incubated in a fixation/permeabilization solution (Cat #: 88-8824-00; ThermoFisher, Waltham, MA, USA) and stained with a fluorescently conjugated anti-mouse antibody against intracellular FOXP3 (Cat #: 12400-31; SouthernBiotech, Birmingham, AL, USA). All other samples were washed and fixed using 1% paraformaldehyde. Samples were stored at 4 °C until flow cytometric analysis could be performed using a Gallios Flow Cytometer (Beckman Coulter). A minimum of 1 × 104 events per sample were captured and analyzed with Kaluza Analysis 2.1 software (Beckman Coulter).
Wise R.M., Harrison M.A., Sullivan B.N., Al-Ghadban S., Aleman S.J., Vinluan A.T., Monaco E.R., Donato U.M., Pursell I.A, & Bunnell B.A. (2020). Short-Term Rapamycin Preconditioning Diminishes Therapeutic Efficacy of Human Adipose-Derived Stem Cells in a Murine Model of Multiple Sclerosis. Cells, 9(10), 2218.
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3

Quantifying Tumor Immune Infiltrates

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Tumors obtained at necropsy were digested in media containing 1 mg/mL collagenase and 20 μg/mL DNAse I (Sigma) for 2 hours at 37°C, and passed through a 100 μm screen to obtain a single-cell suspension. CD8+ T cells were isolated (STEMCELL Technologies, Vancouver, BC Canada) and then stained with anti-CD3 (17A2, eBioscience), anti-CD8 (53-6.7, eBioscience) and anti-CD69 (H1.2F3, eBioscience) antibodies and DAPI. For PD-L1 quantification, tumor suspensions were stained with anti-CD45 (30-F11, BD Bioscience), anti-CD11b (M1/70, eBioscience), anti-GR1 (1A8, BD Bioscience), anti-F4/80 (BM8.1, Tonbo Biosciences), anti-PD-L1 (MIH5, eBioscience) and DAPI.
For PD-1 quantification on antigen-specific T cells, splenocytes obtained from immunized animals were enriched for CD8+ T cells and stained as above, along with tetramers specific for the SSX2 p41 or p103 epitopes (NIH Tetramer Core Facility, Atlanta GA), anti-PD-1 (J43, BD Bioscience, San Jose CA) and Ghost Dye–780 (Tonbo Bioscience, San Diego, CA).
For in vitro culture studies, cells were treated for 18 hours with 1 μg/mL recombinant mIFNγ (Shenandoah Biotechnology, Warwick PA), or cultured for 48 hours with CD8+ splenocytes isolated from immunized animals, and then collected using non-enzymatic cell dissociation solution (Sigma), and stained as above.
Rekoske B.T., Smith H.A., Olson B.M., Maricque B.B, & McNeel D.G. (2015). PD-1 or PD-L1 Blockade Restores Antitumor Efficacy Following SSX2 Epitope-Modified DNA Vaccine Immunization. Cancer immunology research, 3(8), 946-955.
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4

Discriminating Intravascular and Extravascular Cells

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To discriminate intravascular from extravascular cells, we injected mice i.v. with biotin-conjugated anti-CD8α as described51 (link). Three minutes after the injection, we sacrificed the mice and harvested tissues as described52 (link). Isolated cells were stained with antibodies to CD45.1 (A20), CD8α (53–6.7), CD8β (YTS156.7.7), CD27 (LG.3A10), CD62L (MEL-14), Ly6C (AL-21 and HK1.4), CD127 (A7R34), CCR9 (CW-1.2), CD44 (IM7), CD69 (H1.2F3), CD103 (M290 or 2E7), CD90.1 (OX-7 or His51), CD122 (TM-β1), CD49a (Ha31/8), CX3CR1 (SA011F11), α4β7 (DATK32) and KLRG1 (2F1), all from BD Biosciences, Tonbo Biosciences, Biolegend or Affymetrix eBiosciences. LCMV-specific T cells were stained with fluorescently conjugated H-2Db/gp33 MHC I tetramers. Ova-specific T cells were stained with fluorescently conjugated H-2Kb/SIINFEKL MHC I tetramers. Endogenous VSV-specific cells were stained with fluorescently conjugated H-2Kb/N MHC I tetramers. Allophycocyanin (APC) or Phycoerythrin (PE)-conjugated anti-Granzyme B (GB12 or GB11, Invitrogen) antibody intracellular staining was performed using the Cytofix/Cytoperm kit (BD Pharmigen) following manufacturer’s instructions. Cell viability was determined with Ghost Dye 780 (Tonbo Biosciences). The stained samples were acquired on LSRII or LSR Fortessa flow cytometers (BD) and analyzed with FlowJo software (Treestar).
Fonseca R., Beura L.K., Quarnstrom C.F., Ghoneim H.E., Fan Y., Zebley C.C., Scott M.C., Fares-Frederickson N.J., Wijeyesinghe S., Thompson E.A., da Silva H.B., Vezys V., Youngblood B, & Masopust D. (2020). Developmental plasticity allows outside-in immune responses by resident memory T cells. Nature immunology, 21(4), 412-421.
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5

Discriminating Intravascular and Extravascular Cells

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To discriminate intravascular from extravascular cells, we injected mice i.v. with biotin-conjugated anti-CD8α as described51 (link). Three minutes after the injection, we sacrificed the mice and harvested tissues as described52 (link). Isolated cells were stained with antibodies to CD45.1 (A20), CD8α (53–6.7), CD8β (YTS156.7.7), CD27 (LG.3A10), CD62L (MEL-14), Ly6C (AL-21 and HK1.4), CD127 (A7R34), CCR9 (CW-1.2), CD44 (IM7), CD69 (H1.2F3), CD103 (M290 or 2E7), CD90.1 (OX-7 or His51), CD122 (TM-β1), CD49a (Ha31/8), CX3CR1 (SA011F11), α4β7 (DATK32) and KLRG1 (2F1), all from BD Biosciences, Tonbo Biosciences, Biolegend or Affymetrix eBiosciences. LCMV-specific T cells were stained with fluorescently conjugated H-2Db/gp33 MHC I tetramers. Ova-specific T cells were stained with fluorescently conjugated H-2Kb/SIINFEKL MHC I tetramers. Endogenous VSV-specific cells were stained with fluorescently conjugated H-2Kb/N MHC I tetramers. Allophycocyanin (APC) or Phycoerythrin (PE)-conjugated anti-Granzyme B (GB12 or GB11, Invitrogen) antibody intracellular staining was performed using the Cytofix/Cytoperm kit (BD Pharmigen) following manufacturer’s instructions. Cell viability was determined with Ghost Dye 780 (Tonbo Biosciences). The stained samples were acquired on LSRII or LSR Fortessa flow cytometers (BD) and analyzed with FlowJo software (Treestar).
Fonseca R., Beura L.K., Quarnstrom C.F., Ghoneim H.E., Fan Y., Zebley C.C., Scott M.C., Fares-Frederickson N.J., Wijeyesinghe S., Thompson E.A., da Silva H.B., Vezys V., Youngblood B, & Masopust D. (2020). Developmental plasticity allows outside-in immune responses by resident memory T cells. Nature immunology, 21(4), 412-421.
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6

Dengue Virus Infection Immunostaining Protocol

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Cells were washed with PBS containing 2% FBS, and stained with Ghost Dye 780 (Tonbo Biosciences, San Diego, CA) at 4 ℃ for 30 min. Cells were then fixed with 4% para-formaldehyde, and permeabilized with 0.1% saponin at 4 ℃ for 15 min. The infected cells were incubated with mouse 4G2 (anti-DENV E protein) antibody for 2 h and Alexa-Fluor-488-conjugated goat anti-mouse IgG antibody at a dilution of 1:40 at 4 ℃ for 1 h. The cells were washed and analyzed with LSR II flow cytometer (BD Bioscience, San Jose, CA) using FlowJo software (Tree Star, San Carlos, CA).
Phumesin P., Panaampon J., Kariya R., Limjindaporn T., Yenchitsomanus P.T, & Okada S. (2022). Cepharanthine inhibits dengue virus production and cytokine secretion. Virus Research, 325, 199030.
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7

Retinal Immune Cell Profiling

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Both retinas from each mouse were dissected, pooled, and digested with 1.5 mg/mL collagenase A (Sigma-Aldrich) for 30 min at 37 °C in a water bath. Digestion was stopped by adding PBS−10% FBS, the retinas were mashed through a 100-µm cell strainer, and the cell suspension was then subjected to flow cytometry staining. To exclude dead cells from the analysis, samples were stained with Ghost Dye 780 (Tonbo Biosciences, San Diego, CA, USA), following the protocol recommended by the manufacturer. Next, the samples were treated with Fc block (obtained from the supernatant of the 2.4G2 hybridoma) prior to surface staining with the following antibodies: anti-CD45-eFluor450 (clone 30-F11, Biolegend, San Diego, CA, USA); anti-CD11b-PerCPCy5.5 (clone M170, Biolegend); anti-F4/80-APC (clone BM8, eBioscience, San Diego, CA, USA), anti-I-A/I-E-PE (clone M5/114.15.2, Biolegend); anti-Ly6c-biotin (clone HK1.4, Beckman Coulter, Indianapolis, IN, USA); and anti-CD11c-FITC (clone HL3BD, BD Pharmingen, San Diego, CA, USA). All samples were incubated with streptavidin-Brilliant Violet 605 (Biolegend) for anti-Ly6c-biotin detection.
Events were acquired using an FACS Canto II cell analyzer (Becton Dickinson, Franklin Lakes, NJ, USA), and all cell doublets and dead cells were excluded from the analysis. Data analysis was performed using FlowJo v10 software (Tree Star, Ashland, OR, USA).
Sánchez-Cruz A., Méndez A.C., Lizasoain I., de la Villa P., de la Rosa E.J, & Hernández-Sánchez C. (2021). Tlr2 Gene Deletion Delays Retinal Degeneration in Two Genetically Distinct Mouse Models of Retinitis Pigmentosa. International Journal of Molecular Sciences, 22(15), 7815.
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8

Identifying Intravascular CD8+ T Cells

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To discriminate intravascular from extravascular cells, we injected mice i.v. with BV605-conjugated anti-CD8α antibody and after 3 min, mice were sacrificed and tissues were harvested. Lymphocytes were isolated as described (9 , 10 ) and stained with antibodies to CD8α (53–6.7), CD62L (MEL-14), Ly6C (HK1.4), CD127 (A7R34), CD44 (IM7), CD69 (H1.2F3), CD103 (M290) all from BD Biosciences, Tonbo Biosciences, Biolegend or Affymetrix eBiosciences and H2-Db/N219–227 MHC (major histocompatibility complex) I tetramers (made in-house) and ghost dye 780 (Tonbo Biosciences). For monomer preparation and all peptide-dependent assays described below, the N219–227 peptide sequence was LALLLLDRL. Human PBMCs were stained with CD3ε (SP34–2), CD4 (OKT4), CD8 (SK1), IFNγ (B27) and TNFα (MAb11). Stained samples were acquired on LSRII or LSR Fortessa flow cytometers (BD Biosciences) and analyzed with FlowJo software (BD Biosciences). Cells were gated on singlets, live lymphocytes, CD8 T cells and/or tetramer-specific cells as indicated.
Joag V., Wijeyesinghe S., Stolley J.M., Quarnstrom C.F., Dileepan T., Soerens A.G., Sangala J.A., O’Flanagan S.D., Gavil N.V., Hong S.W., Bhela S., Gangadhara S., Weyu E., Matchett W., Thiede J., Krishna V., Cheeran M.C., Bold T., Amara R., Southern P., Hart G.T., Schifanella L., Vezys V., Jenkins M.K., Langlois R.A, & Masopust D. (2021). Mouse SARS-CoV-2 epitope reveals infection and vaccine-elicited CD8 T cell responses. Journal of immunology (Baltimore, Md. : 1950), 206(5), 931-935.
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9

Isolation and Characterization of Hepatic Stellate Cells

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HSCs were enriched as described previously (5 ). Cell suspensions were blocked in FACS Buffer (0.5% FCS, 0.01% Sodium azide, 1X PBS) supplemented with 10% normal mouse serum (Sigma) and anti-CD16/32 (2.4G2) then stained for CD45.2 (104; Tonbo Biosciences, CA). Dead cells were excluded using standard staining procedures for live/dead with Ghost Dye 780 (Tonbo) and fixed with BD Cytofix/Cytoperm (BD Biosciences) according to manufacturer’s instructions. To determine activation, intracellular α-SMA-PE (R&D systems) was stained at room temperature. HSCs were acquired on a FACSAria II equipped with a UV laser (355nm) (BD Biosciences) and were defined as UV auto-fluorescent positive and CD45-negative populations as indicated in Supplemental Figure S1C. Auto-fluorescence and debris were excluded using a 405nm laser (450/50BP).
Tedesco D., Thapa M., Gumber S., Elrod E.J., Rahman K., Ibegbu C.C., Magliocca J.F., Adams A.B., Anania F, & Grakoui A. (2016). CD4+ Foxp3+ T cells promote aberrant IgG production and maintain CD8+ T cell suppression during chronic liver disease. Hepatology (Baltimore, Md.), 65(2), 661-677.
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10

Intravascular T Cell Labeling Protocol

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We repurposed an intravascular cell labeling technique, as described previously39 (link), to assess availability of antibody to vascular and parenchymal T cells. Briefly, mice were injected intravenously with biotin-conjugated anti-CD8α. Three minutes after the injection, mice were euthanized and tissues were harvested as described10 (link),36 (link). In brief, FRT tissue was removed, digested in Collagenase IV (Sigma) with DNAse for 1 hour, then dissociated via gentleMACS Dissociator (Miltenyi Biotec), and lymphocytes were purified on a 44/67% Percoll (GE Healthcare) gradient. Isolated cells were stained with Steptavidin-BV650 (Biolegend) to label biotin-CD8α bound cells, and antibodies to CD44 (clone IM7, BioLegend), CD45.1 (clone A20, Biolegend), Thy1.1 (Clone OX7, Biolegend), and CD8β (clone Ly-3, Biolegend). All cells were stained at antibody dilutions of 1:100 except Thy1.1, which was 1:1000. Cell viability was determined with Ghost Dye 780 (Tonbo Biosciences). The stained samples were acquired with LSRII or LSR Fortessa flow cytometers (BD Biosciences) and analyzed with FlowJo software (Treestar).
Frolov I., Hoffman T.A., Prágai B.M., Dryga S.A., Huang H.V., Schlesinger S, & Rice C.M. (1996). Alphavirus-based expression vectors: strategies and applications.. Proceedings of the National Academy of Sciences of the United States of America, 93(21), 11371-11377.
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