Ghost dye uv450

Manufactured by Cytek Biosciences

The Ghost Dye UV450 is a fluorescent stain used for the detection and analysis of cells in flow cytometry applications. It is designed to bind to nucleic acids, providing a specific signal that can be detected using ultraviolet (UV) excitation and a 450 nm emission filter.

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Lab products found in correlation

Cytofix cytoperm kit, by BD (2 mentions) Lsrfortessa, by BD (2 mentions) Brefeldin a golgiplug, by BD (1 mentions) Red 780, by Cytek Biosciences (1 mentions) Ionomycin, by Merck Group (1 mentions) Macsquant analyzer, by Miltenyi Biotec (1 mentions) Fc receptor block, by BD (1 mentions) 5 laser lsr 2, by BD (1 mentions) Foxp3 fix perm solution, by BioLegend (1 mentions) Ghost dye violet 510, by Cytek Biosciences (1 mentions) Ethylene diamine tetraacetic acid (edta), by Thermo Fisher Scientific (1 mentions) Bovine serum albumin (bsa), by Gemini Bio (1 mentions) Facsdiva, by BD (1 mentions) Hepes, by Corning (1 mentions) Anti ifn γ xmg1, by BioLegend (1 mentions) Monensin, by Thermo Fisher Scientific (1 mentions) Anti cd45 30 f11, by Thermo Fisher Scientific (1 mentions) Ic fixation buffer, by Thermo Fisher Scientific (1 mentions) Anti tcrβ h57 597, by Thermo Fisher Scientific (1 mentions) Anti il 10 jes5 16e3, by Thermo Fisher Scientific (1 mentions)

13 protocols using ghost dye uv450

Multi-Dimensional Single-Cell Phenotyping

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Single cell suspensions were prepared from isolated tissues. Samples of 1 × 10⁶ cells were stained with anti-mouse antibodies conjugated with following fluorochromes: BV421, BV510/V500, FITC, PE, PerCP-Cy5.5/PerCP-eF710, PE-Cy7, APC, APC Cy7/APC-eF780, BV650/SB645, BV785. Surface staining was performed using antibodies against F4/80, Ly6G, CD3, CD4, CD11b, CD44, CCR6, CXCR3, CCR7, and S1PR1. Ghost Dye UV 450 or Red 780 (Tonbo, CA) were used to exclude dead cells. Intracellular staining was performed after stimulating cells with 50 ng/ml PMA and 1 µg/ml Ionomycin (MilliporeSigma, MA) in the presence of Brefeldin A (Golgi Plug, BD Biosciences, CA) for 4–6 hr at 37°C in cell culture media. Following the surface and viability staining, cells were fixed with 4% paraformaldehyde (PFA) and permeabilized with Triton buffer (0.5% Triton X-100% and 0.1% BSA in PBS). Fluorochrome-conjugated (see above) anti-mouse Foxp3, IL-17A, and IFN-γ were used for intracellular staining. Cells were acquired on BD LSR Fortessa (BD Biosciences, CA), MACSQuant Analyzer (Miltenyi Biotec, Germany), or Cytoflex (Beckham Coulter, CA). Fcs files were analyzed using FlowJo (FlowJo LLC, OR).

Wei J., Mattapallil M.J., Horai R., Jittayasothorn Y., Modi A.P., Sen H.N., Gronert K, & Caspi R.R. (2020). A novel role for lipoxin A4 in driving a lymph node–eye axis that controls autoimmunity to the neuroretina. eLife, 9, e51102.

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Multiparameter Flow Cytometry Protocol

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Three million cells per sample were added to BD Falcon 5 mL polypropylene round-bottom tubes. Cells were washed with 2 mL of 1X PBS and centrifuged at 1200 rpm for 5 minutes at 4°C. Supernatant was removed and cells were washed in 2 mL of FACS buffer (1X PBS + 3% FBS). Supernatant was removed and 3 μL of Fc Receptor Block (BD) was diluted in 50 μL of FACS buffer per sample and incubated for 15 minutes at 4°C. Cells were washed with 2 mL of FACS buffer and centrifuged at 1200 rpm for 5 minutes at 4°C. Supernatant was removed and antibodies were diluted in 50 μL of FACS buffer and incubated for 30 minutes at 4°C. Cells were washed with 2 mL of FACS buffer and centrifuged at 1200 rpm for 5 minutes at 4°C. If biotinylated antibody was used, streptavidin was diluted in 100 μL FACS buffer per sample and incubated for 30 minutes at 4°C. Viability staining was performed by adding Tonbo Ghost Dye UV450 (1:500) or DAPI (1:1000). Cells were analyzed on the 5-Laser BD LSR II or sorted on the 5-Laser FACS Aria III in the VUMC Flow Cytometry Shared Resources.

Meyer A.R., Engevik A.C., Madorsky T., Belmont E., Stier M.T., Norlander A.E., Pilkinton M.A., McDonnell W.J., Weis J.A., Jang B., Mallal S.A., Peebles RS J.r, & Goldenring J.R. (2020). Group 2 innate lymphoid cells coordinate damage response in the stomach. Gastroenterology, 159(6), 2077-2091.e8.

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PyMT Peptide MHC-I Tetramer Staining

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The three PyMT peptides folded into MHC-I tetramers with the haplotype H-2Dq with human β₂M and conjugated to R-PE were kindly provided by the NIH Tetramer Core Facility (Emory University). Whole splenocytes were obtained from tumor-bearing mice as described above, and flow cytometry staining was performed. Splenocytes were first stained with the fixable viability dye (Ghost Dye UV450; Tonbo Biosciences) for 30 minutes on ice to exclude dead cells. Cells were then stained with the tetramer pool containing all three tetramers (1:100 dilution each) for 30 minutes at room temperature. Mouse FcR blocking was performed by incubating with anti-CD16/CD32 for 10 minutes on ice followed by surface marker staining for 30 minutes on ice with a panel of antibodies including CD3e (APC, clone 145–2C11), CD4 (violetFluor450, clone GK1.5), CD8a (FITC, clone 53–6.7), CD62L (PE-Cy7, clone MEL-14), and CD44 (redFluor710, clone IM7). In addition, for compensation purposes in the tetramer-PE channel, anti-CD3e conjugated with PE was used. Data acquisition and analysis were performed as described above (see flow cytometry section).

Lai S.C., Gundlapalli H., Ekiz H.A., Jiang A., Fernandez E, & Welm A.L. (2021). Blocking Short-Form Ron Eliminates Breast Cancer Metastases through Accumulation of Stem-Like CD4+ T Cells That Subvert Immunosuppression. Cancer Discovery, 11(12), 3178-3197.

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Comprehensive Immune Cell Isolation and Profiling

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Spleens and liver-draining lymph nodes were collected from humanely euthanized mice between 8–12 weeks of age and homogenized through a 70 μm cell strainer, then further suspended using a 27-gauge needle. Liver non-parenchymal cells were isolated by two-step perfusion as described in “primary hepatocyte isolation and culture”. Hepatocytes were removed from the cell suspension by sequential centrifugation at 50xg; the supernatants containing NPCs were centrifuged at 300xg for 5 minutes to pellet the cells. Red blood cells were removed from NPCs, splenocytes, and lymph node suspensions by incubating in ACK lysis buffer for 5 minutes at room temperature. Cleared cells were pelleted by centrifugation at 500 g for 5 minutes, then washed twice in phosphate-buffered saline (PBS).
Surface marker staining was performed as follows: First, viability staining was performed for 20 minutes on ice in PBS with Tonbo Ghost Dye UV450, with all samples protected from light through this and subsequent steps. Conjugated antibodies were added and cells were incubated 15–20 minutes on ice. See Supplemental File 1 for antibody panels used. Cells were washed 3 times in staining buffer (PBS with 5% FBS and 0.1% sodium azide).
Intracellular staining was performed using the Foxp3 Fix/Perm solution (BioLegend) according to manufacturer’s instructions.

Hanley K.L., Liang Y., Wang G., Lin X., Yang M., Karin M., Fu W, & Feng G.S. (2022). Concurrent Disruption of Ras/MAPK and NF-κB Pathways Induces Circadian Deregulation and Hepatocarcinogenesis. Molecular cancer research : MCR, 20(3), 337-349.

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Murine Spleen Cell Isolation and Characterization

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Spleens were processed via mechanical dissociation, and RBCs were lysed with ammonium-chloride-potassium buffer (in house). Live/dead discrimination was performed using GhostDye Violet 510 or GhostDye UV450 (Tonbo Biosciences). Surface staining was performed in ice-cold PBS (Cytiva) + 0.5% BSA (GeminiBio) + 2.5% HEPES (Corning) + 2.5 mM EDTA (Fisher Bioreagents). Cells were stained in the presence of FcR blocking Ab 2.4G2 (in-house). Intracellular staining was performed using the BD Biosciences Cytofix/Cytoperm kit. Data were collected using an LSRII (BD Biosciences) with FACSDiva (BD Biosciences) and analyzed using FlowJo software (Tree Star Inc.).

Cosgrove H.A., Gingras S., Kim M., Bastacky S., Tilstra J.S, & Shlomchik M.J. (2023). B cell–intrinsic TLR7 expression drives severe lupus in TLR9-deficient mice. JCI Insight, 8(16), e172219.

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Cytokine Analysis of CD4+ T Cells

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Single cell suspension of PECs and LNs were stained with anti-CD45 (30-F11: eBioscience; San Diego, CA), anti-CD4 (RM4–5: Biolegend; San Diego, CA), and anti-TCRβ (H57–597: eBioscience) antibodies. Subsequently, the cells were stimulated with cell stimulation cocktail (eBioscience) and monensin (eBioscience) for 5 hours. After stimulation, the cells were stained with Ghost Dye UV450 (TONBO) to identify dead cells. After fixation and permeabilization by IC Fixation Buffer (eBioscience), intracellular cytokines were stained by anti-IFN-γ (XMG1.2: Biolegend), and anti-IL-10 (JES5–16E3: eBioscience) antibodies. Cytokine⁺ CD4⁺ T cells numbers were analyzed by flow cytometry.

Kobiyama K., Vassallo M., Mitzi J., Winkels H., Pei H., Kimura T., Miller J., Wolf D, & Ley K. (2018). A clinically applicable adjuvant for an atherosclerosis vaccine. European journal of immunology, 48(9), 1580-1587.

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MCMV Infection and Antibody Response

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B6 TECs, BALB/c TECs or BALB/c 3T3 cells (American Type Culture Collection, Manassas VA) were infected with MCMV-GFP virus at a multiplicity of infection (MOI) of 1 for each cell type. At 72 hours post infection, cells were detached and incubated with either MCMV− or MCMV+ B6 sera, or with transplant sera (D−/R− or D+/R+) at 1:20 dilution for 1 hour on ice. MCMV uninfected cells were also incubated with sera. Cells were washed and stained for viability (Ghost Dye UV450, TONBO Biosciences, San Diego CA), EpCAM-PE (CD326, Clone G8.8, Biolegend, San Diego CA), and anti-mouse IgG1-APC (Clone A85–1, BD Biosciences, San Jose CA). Samples were analyzed using a LSRFortessa (BD Biosciences) and FlowJo v10 software (TreeStar Inc, Ashland OR). Experiments were performed 3 times independently.

Saunders U., Li M., Boddeda S.R., Maher S., Ghere J., Kaptsan I., Dhital R., Velazquez V., Guo L., Chen B., Zeng Q., Schoeb T.R., Cianciolo R, & Shimamura M. (2021). Murine Cytomegalovirus-Induced Complement-fixing Antibodies Deposit in Murine Renal Allografts During Acute Rejection. Transplantation, 105(8), 1718-1729.

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Th17 Cell Differentiation and Analysis

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Three days after Th17 cells differentiation, cells were restimulated with 50 ng/ml PMA, 1 μM ionomycin, and 0.07% of Golgi Stop for 4 h. Following restimulation, cells were stained with viability dye (Ghost Dye UV 450; Tonbo Biosciences), blocked with an anti-FcR Ab (clone 2.4G2), and surface stained with APC-Cy7 anti-CD3 (clone 145-2C11), FITC anti-CD4 (clone GK1.5), BV786 anti-CD90.2 (clone 53-2.1), and APC anti-IL-23R (clone 12B2B64). Cells were then fixed, permeabilized using the Foxp3/transcription factor staining kit (Tonbo Biosciences), and intracellularly stained PE-Cy7 anti–IL-17A (clone eBio17B7). Flow cytometry analysis was conducted on LSR II flow cytometer, and all data were processed using FlowJo software version 10. Antibodies were purchased from Thermo fisher (Invitrogen). In select experiments, Cell Tracer Violet dye was added per manufacturer's instructions (ThermoFisher) prior to Th17 cell differentiation. Th17 cell proliferation was determined by flow cytometry 3 days later by gating on viable, CD4+ IL-17A+ cells and measuring the proliferation index using FlowJo software.

Fuseini H., Cephus J.Y., Wu P., Davis J.B., Contreras D.C., Gandhi V.D., Rathmell J.C, & Newcomb D.C. (2019). ERα Signaling Increased IL-17A Production in Th17 Cells by Upregulating IL-23R Expression, Mitochondrial Respiration, and Proliferation. Frontiers in Immunology, 10, 2740.

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DENV Envelope Protein Detection in UT-7 Cells

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UT-7 cells were stained with Ghostdye UV450 (Tonbo) per the manufacturer’s instructions. Cells were blocked with normal mouse serum (Invitrogen) for 10 minutes at room temperature. Then, cells were fixed and permeabilized using the Cytofix/Cytoperm kit (BD Biosciences) according to the manufacturer’s instructions. Samples were then incubated for 30 minutes at 4°C with antibody against DENV envelope (4G2, FITC, Millipore). Samples were run on an LSRII cytometer (BD) and data were analyzed using FlowJo (TreeStar). Viability was also assessed via trypan blue exclusion. A sample of infected UT-7 cells were collected every other day post infection and diluted in cell culture media with trypan blue (final dilution 1:10). Total cells, trypan blue stained cells, and unstained cells were counted via hemocytometer. Viable cells were those that were unstained by trypan blue. The percentage of viable cells was calculated by dividing the number of unstained cells by the number of total cells.
Gating strategies for all flow cytometry assays are described in S1 Text. All data are available at the flow cytometry repository: https://flowrepository.org/id/FR-FCM-Z2B4

Vogt M.B., Lahon A., Arya R.P., Spencer Clinton J.L, & Rico-Hesse R. (2019). Dengue viruses infect human megakaryocytes, with probable clinical consequences. PLoS Neglected Tropical Diseases, 13(11), e0007837.

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Comprehensive Immune Cell Profiling by Flow Cytometry

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PBMCs were stained and analyzed by flow cytometry as previously described.38 (link) The following antibodies were used for cell surface staining: FITC-conjugated antihuman CD45 (Biolegend, Catalog #368508), PerCP-Cyanine5.5-conjugated antihuman CD3 (Tonbo Biosciences, Catalog #65–0037), PE-conjugated antihuman CD4 (Tonbo, Catalog #50–0048), APC-conjugated antihuman CD8 (Tonbo, Catalog # 20–0087), Brilliant Violet 421-conjugated antihuman CD279 (PD-1, Biolegend, Catalog #329920). Ghost Dye UV 450 (Tonbo) was used to assess live versus dead status of cells. Samples were acquired on a LSRII analyzer (BD Biosciences) and analyzed with FlowJo software (Treestar). For all FACS, experiments debris and dead cells were excluded from the analyzed gates.

Dorff T., Hirasawa Y., Acoba J., Pagano I., Tamura D., Pal S., Zhang M., Waitz R., Dhal A., Haynes W., Shon J., Scholz M., Furuya H., Chan O.T., Huang J, & Rosser C. (2021). Phase Ib study of patients with metastatic castrate-resistant prostate cancer treated with different sequencing regimens of atezolizumab and sipuleucel-T. Journal for Immunotherapy of Cancer, 9(8), e002931.

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