ghost dye red 780 viability dye, by Cytek Biosciences - Product details

1

Quantifying Immune Cell Profiles in Intracranial Melanoma

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Flow cytometry was used to analyze immune cell populations (12 (link), 13 (link), 44 (link)) in a minimum of three samples in two independent animal studies. Dissected intracranial melanoma tumors were physically dissociated and filtered through a 70-μm cell filter. Single cells were labeled with primary antibody. Cells without primary antibody labeling were used as unlabeled negative controls; fluorescent beads (UltraComp eBeads^™, Invitrogen^™, Carlsbad, CA) were used as positive/calibration controls and to determine compensation between fluorescent channels. Forward- and side-scatter gating identified single cells and viable cell (Ghost Dye^™ Red 780 Viability Dye, 1:100; Tonbo Biosciences, San Diego, CA) exclusion identified live cells. Fluorescence minus one (FMO) methodology was used to determine gating. Flow cytometry was performed on an Attune NxT Flow Cytometer (Thermo Fisher Scientific^™ Inc., Waltham, MA), and compensation matrix computed and data analyzed using FlowJo version 9 software (Ashland, OR) following published flow cytometry guidelines (45 (link)).
Antibodies used were: CD4 (BV510, 1:400, clone RM4–5), CD8 (PerCP-cy5.5, 1:200, clone 53–6.7), FOXP3 (BV421, 1:100, clone MF-14), all acquired from BioLegend^® Inc. (San Diego, CA); and CD45 (PE-cy7, 1:200, clone 30-F11), CD3 (FITC, 1:200, clone 17A2), both acquired from Tonbo Biosciences.

Clark P.A., Sriramaneni R.N., Bates A.M., Jin W.J., Jagodinsky J.C., Hernandez R., Le T., Jeffery J.J., Marsh I.R., Grudzinski J.J., Aluicio-Sarduy E., Barnhart T.E., Anderson B.R., Chakravarty I., Arthur I.S., Kim K., Engle J.W., Bednarz B.P., Weichert J.P, & Morris Z.S. (2021). Low-Dose Radiation Potentiates the Propagation of Anti-Tumor Immunity against Melanoma Tumor in the Brain after In Situ Vaccination at a Tumor outside the Brain. Radiation research, 195(6), 522-540.

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2

Multiparametric flow cytometry analysis of BAL cells

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BAL cell samples were incubated for 10 min at room temperature with Human TruStain FcX and Ghost Dye Red 780 viability dye (Tonbo Biosciences, San Diego, CA, USA) before staining with CD4 PerCP-Cy5.5 (clone L200, BD Biosciences, Mississauga, ON, Canada), CD45RA PE-CF594 (clone 5H9, BD Biosciences), CD3 Alexa Fluor 700 (clone SP34-2, BD Biosciences), CCR7 BV711 (clone G043H7, BioLegend, San Diego, CA, USA), CD8 BV786 (clone RPA-T8, BD Biosciences), CD45 BUV395 (clone D058-1283, BD Biosciences). Erythrocytes were removed via incubation with 1× BD Biosciences FACS Lysing solution before fixation and permeabilization of the remaining cells with BD Biosciences Cytofix/Cytoperm solution and removal from CL-4. Following removal from containment, all samples were examined using a BD Biosciences FACSymphony A5 flow cytometer; fluorescence spillover was corrected for using singly stained compensation beads (Invitrogen UltraComp eBeads or ArC Amine Reactive Beads). Data were analyzed using FlowJo version 10 software.

Warner B.M., Chan M., Tailor N., Vendramelli R., Audet J., Meilleur C., Truong T., Garnett L., Willman M., Soule G., Tierney K., Albietz A., Moffat E., Higgins R., Santry L.A., Leacy A., Pham P.H., Yates J.G., Pei Y., Safronetz D., Strong J.E., Susta L., Embury-Hyatt C., Wootton S.K, & Kobasa D. (2024). Mucosal Vaccination with a Newcastle Disease Virus-Vectored Vaccine Reduces Viral Loads in SARS-CoV-2-Infected Cynomolgus Macaques. Vaccines, 12(4), 404.

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Neutrophil Isolation from Lung Tissue

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Lung tissues were collected and subjected to collagenase digestion (200 U/ml collagenase type I, Worthington Biochemical Corporation, Lakewood, NJ, USA) in Roswell Park Memorial institute 1640 (RPMI) medium containing 1% penicillin/streptomycin, 0.05% DNase I (all from Thermo Fisher Scientific, Waltham, MA, USA), and 1% HEPES (Merck) at 37°C for 1 h. The reaction was stopped using a complete RPMI medium containing 5% fetal bovine serum (FBS, Thermo Fisher Scientific). The single suspended cells were stained with eFluor 506 antimouse CD45 antibody (Thermo Fisher Scientific), Ghost dye™ Red 780 viability Dye (TONBO Biosciences, San Diego, CA, USA), and PE antimouse Ly-6G/Ly-6C (Gr-1) antibody (Biolegend, San Diego, CA, USA) to detect the neutrophil population and then analyzed by flow cytometry (Attune NxT Flow Cytometer, Thermo Fisher Scientific).

Ting N.C., Chen Y.H., Chen J.C., Huang W.C., Liou C.J., Chen L.C., Yang S.H, & Kuo M.L. (2022). Perilla Fruit Water Extract Attenuates Inflammatory Responses and Alleviates Neutrophil Recruitment via MAPK/JNK-AP-1/c-Fos Signaling Pathway in ARDS Animal Model. Evidence-based Complementary and Alternative Medicine : eCAM, 2022, 4444513.

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4

Flow Cytometric Analysis of Adipose B Cells

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Isolated cells were incubated with Ghost Dye Red 780 Viability Dye (TONBO biosciences) for 30 minutes on ice, in the dark, then washed and stained with FcBlock and surface antibodies for 45 minutes to 12 hours at 4°C in the dark. Antibodies used are documented in Table S1. For intracellular staining of cytokines, cells were treated with GolgiStop (containing monensin) for 4 hours and fixed for 20 minutes using fixation/permeabilization solution (BD, 554715) prior to intracellular staining for 45 minutes. Analysis was performed on a BD Fortessa X-30 and BD FACSSymphony A3 cytometers and using FlowJo v10; gating schemes are shown in relevant supplementary figures. Antibodies used can be found in Table S1. For fluorescence associated cell sorting: 15,000 live CD45⁺ CD19⁺ CD11b⁺ and live CD45⁺ CD19⁺ CD11b⁻ were sorted on a BD FACS Aria II into RPMI with 40% FBS. Cells were pelleted and resuspended in RNA lysis buffer (Invitrogen; 46-6001) for RNA isolation. Adipose B cells were pooled from 2 mice per sample to account for biological variability.

Carey A., Nguyen K., Kandikonda P., Kruglov V., Bradley C., Dahlquist K.J., Cholensky S., Swanson W., Badovinac V.P., Griffith T.S, & Camell C.D. (2024). Age-associated accumulation of B cells promotes macrophage inflammation and inhibits lipolysis in adipose tissue during sepsis. Cell reports, 43(3), 113967.

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5

Phenotyping Monocyte Subsets by Flow Cytometry

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Monocyte controls (untreated), EEMos, and LPS-EEMos were collected and counted with a Beckman Coulter Z1 Particle Counter and then 1 × 10⁶ cells were incubated with Fc block (BD Pharmingen, San Jose, CA, USA, cat#: 564220) for 10 min at room temperature. Cells were then stained at 4 °C for 20–30 min with anti-human antibodies in staining buffer (PBS with 2% FBS). All antibodies were purchased from BioLegend (San Diego, CA). Antibodies included CD206 (15-2, cat# 321105), CD163 (GHI/61, cat# 333617), PD-L1 (29E.2A3, cat# 329721), PD-L2 (24F.10C12, cat# 329608), CD14 (HCD14, cat# 325627), CD16 (3G8, cat# 302025), HLA-DR (L243, cat# 307639), CD73 (TY/11.8, cat# 127223), and CD86 (IT2.2, cat# 305431). The cells were washed with PBS, centrifuged at 300g for 10 min, and Ghost Dye™ Red 780 viability dye (Tonbo Biosciences, cat# 13-0865) was added for 20 min at room temperature. Cells were then washed with staining buffer, spun down, and resuspended in 400 μL of staining buffer. Cells were then run on an Attune™ NXT flow cytometer (Thermo Fisher Scientific). Subsequent analysis was performed using Flowjo™ 9.9.6 software (BD).

Forsberg M.H., Kink J.A., Thickens A.S., Lewis B.M., Childs C.J., Hematti P, & Capitini C.M. (2021). Exosomes from primed MSCs can educate monocytes as a cellular therapy for hematopoietic acute radiation syndrome. Stem Cell Research & Therapy, 12, 459.

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6

Quantifying Complement-Mediated C3b Deposition

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Endothelial cells (50,000 cells per well) were seeded in a 96-well plate in serum dilution buffer (SDB) (1× annexin V buffer (51-66121E, BD Pharmingen), 1 mM MgCl₂ (68475, Sigma) and 1% BSA (A9576, Sigma)). Pooled normal human serum (NHS, Complement Technology) or cynomolgus monkey serum (NHP01SRM, BioIVT) diluted in SDB were added to appropriate wells at a final concentration of 25% and incubated for 30 min at 37 °C. For negative controls, cells were treated with 25% serum containing 10 mM EDTA (15575038, Thermo Fisher) to inactivate complement. After incubation, cells were washed and stained with phycoerythrin-conjugated anti-C3b antibodies at a 1:100 dilution (846104, BioLegend) and Ghost Dye Red 780 viability dye at a 1:500 dilution (13-0865, Tonbo Biosciences) for 30 min at 4 °C in the dark. Cells were washed twice, immediately acquired on a BD FACSymphony A3 cytometer and analysed in Flow Jo. C3b deposition was plotted as MFI and statistics were calculated using GraphPad Prism v8.2.0.

Anand R.P., Layer J.V., Heja D., Hirose T., Lassiter G., Firl D.J., Paragas V.B., Akkad A., Chhangawala S., Colvin R.B., Ernst R.J., Esch N., Getchell K., Griffin A.K., Guo X., Hall K.C., Hamilton P., Kalekar L.A., Kan Y., Karadagi A., Li F., Low S.C., Matheson R., Nehring C., Otsuka R., Pandelakis M., Policastro R.A., Pols R., Queiroz L., Rosales I.A., Serkin W.T., Stiede K., Tomosugi T., Xue Y., Zentner G.E., Angeles-Albores D., Chris Chao J., Crabtree J.N., Harken S., Hinkle N., Lemos T., Li M., Pantano L., Stevens D., Subedar O.D., Tan X., Yin S., Anwar I.J., Aufhauser D., Capuano S., Kaufman D.B., Knechtle S.J., Kwun J., Shanmuganayagam D., Markmann J.F., Church G.M., Curtis M., Kawai T., Youd M.E, & Qin W. (2023). Design and testing of a humanized porcine donor for xenotransplantation. Nature, 622(7982), 393-401.

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7

Quantifying Complement-Mediated C3b Deposition

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Endothelial cells (50,000 cells per well) were seeded in a 96-well plate in serum dilution buffer (SDB) (1× annexin V buffer (51-66121E, BD Pharmingen), 1 mM MgCl₂ (68475, Sigma) and 1% BSA (A9576, Sigma)). Pooled normal human serum (NHS, Complement Technology) or cynomolgus monkey serum (NHP01SRM, BioIVT) diluted in SDB were added to appropriate wells at a final concentration of 25% and incubated for 30 min at 37 °C. For negative controls, cells were treated with 25% serum containing 10 mM EDTA (15575038, Thermo Fisher) to inactivate complement. After incubation, cells were washed and stained with phycoerythrin-conjugated anti-C3b antibodies at a 1:100 dilution (846104, BioLegend) and Ghost Dye Red 780 viability dye at a 1:500 dilution (13-0865, Tonbo Biosciences) for 30 min at 4 °C in the dark. Cells were washed twice, immediately acquired on a BD FACSymphony A3 cytometer and analysed in Flow Jo. C3b deposition was plotted as MFI and statistics were calculated using GraphPad Prism v8.2.0.

Anand R.P., Layer J.V., Heja D., Hirose T., Lassiter G., Firl D.J., Paragas V.B., Akkad A., Chhangawala S., Colvin R.B., Ernst R.J., Esch N., Getchell K., Griffin A.K., Guo X., Hall K.C., Hamilton P., Kalekar L.A., Kan Y., Karadagi A., Li F., Low S.C., Matheson R., Nehring C., Otsuka R., Pandelakis M., Policastro R.A., Pols R., Queiroz L., Rosales I.A., Serkin W.T., Stiede K., Tomosugi T., Xue Y., Zentner G.E., Angeles-Albores D., Chris Chao J., Crabtree J.N., Harken S., Hinkle N., Lemos T., Li M., Pantano L., Stevens D., Subedar O.D., Tan X., Yin S., Anwar I.J., Aufhauser D., Capuano S., Kaufman D.B., Knechtle S.J., Kwun J., Shanmuganayagam D., Markmann J.F., Church G.M., Curtis M., Kawai T., Youd M.E, & Qin W. (2023). Design and testing of a humanized porcine donor for xenotransplantation. Nature, 622(7982), 393-401.

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8

Monocyte Surface Marker Characterization

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The detection of cell surface markers of monocyte controls (uneducated), flask or bioreactor EEMos and LPS-EEMos were determined by flow cytometry as described [10 (link), 11 (link)]. All antibodies were purchased from BioLegend (San Diego, CA) CD206: (15–2, cat# 321,105), CD163: (GHI/61, cat# 333,617), PD-L1: (29E.2A3, cat# 329,721), PD-L2: (24F.10C12, cat# 329,608), CD14: (HCD14, cat# 325,627), CD16: (3G8, cat# 302,025), HLA-DR: (L243, cat# 307,639), CD73: (TY/11.8, cat# 127,223), and CD86: (IT2.2, cat# 305,431). After a 20-min staining, monocytes were washed with PBS (Hyclone), then treated with Ghost Dye™ Red 780 viability dye (Tonbo Biosciences, San Diego, CA), cat# 13–0865). Stained cells were washed, assayed on an Attune™ NXT flow cytometer (Thermo Fisher Scientific) and analyzed using Flowjo™ 9.96 software (BD Biosciences, San Diego, CA).

Kink J.A., Bellio M.A., Forsberg M.H., Lobo A., Thickens A.S., Lewis B.M., Ong I.M., Khan A., Capitini C.M, & Hematti P. (2024). Large-scale bioreactor production of extracellular vesicles from mesenchymal stromal cells for treatment of acute radiation syndrome. Stem Cell Research & Therapy, 15, 72.

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9

Characterization of CAR T-cell Tumor Infiltration

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Tumors and spleens were removed, mechanically dissociated, and passed through a Corning 35 µm cell strainer. Cell suspensions were centrifuged at 300 g for 10 min, and then digested with ACK lysing buffer (Lonza). The cells were then washed and centrifuged at 300 g for 10 min, and resuspended in 10 mL PBS, 10 µL of which was added to 10 mL of ISOTON diluent and counted on the COULTER COUNTER Z1 Series Particle Counter (Beckman Coulter). From this count, 1×10⁶ cells were added to flow cytometry tubes in staining buffer (PBS with 2% FBS) and stained with antibodies for hCD45, mCD45, scFV 14g2a CAR, and PD-1. The cells were then washed with PBS, centrifuged at 300 g for 10 min, and 0.5 ul of Ghost Dye Red 780 viability dye (Tonbo Biosciences) was added for 20 min at room temperature. Cells were then washed with staining buffer, spun down, and resuspended in 400 µL of staining buffer. Cells were run on an Attune NXT flow cytometer (Thermo Fisher Scientific). Subsequent analyses were performed using FlowJo software (BD). For donor 2, all mice were euthanized on day 25. For donors 1, 3, 4, and 5, spleens and tumors were analyzed as mice reached euthanasia criteria. For donor 6, all mice were euthanized on day 20.

Mueller K.P., Piscopo N.J., Forsberg M.H., Saraspe L.A., Das A., Russell B., Smerchansky M., Cappabianca D., Shi L., Shankar K., Sarko L., Khajanchi N., La Vonne Denne N., Ramamurthy A., Ali A., Lazzarotto C.R., Tsai S.Q., Capitini C.M, & Saha K. (2022). Production and characterization of virus-free, CRISPR-CAR T cells capable of inducing solid tumor regression. Journal for Immunotherapy of Cancer, 10(9), e004446.

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10

Assessing T-cell Viability and Apoptosis

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To assess cell viability 10⁵ cells were incubated with Fas/FasL interaction blocker anti-human Fas mAb (DX2) at 1µg/ml for 1 h, followed by activation in a plate coated with αCD3 (clone HIT3a) (BioLegend) at 10 µg/ml and αCD28 (clone CD28.2) (BD Biosciences) at 3.33 µg/ml (28 (link)). One hour after activation, the apoptosis inducer anti-Fas (clone CH-11) monoclonal Ab (mAb) (Sigma Aldrich) was added at 20 ng/ml. After overnight incubation cells were stained with 0,1 µl Ghost dye Red 780 Viability Dye (Tonbo biosciences) in 100 µl PBS during 30 min at 4°C and with 2 µl APC-conjugated annexin V (No. 640920 Biolegend) in 100 µl Annexin V Binding Buffer (No. 422201, Biolegend) for 15 min at RT, following the manufacturer’s instructions. Data were acquired in a FACSCanto II Analyser Cytometer (405 nm violet laser, 488 nm solid state blue laser and 633 nm He-Ne) (BD Biosciences) and analysed with FlowJo software v10.8.1 (BD Biosciences). The gating strategy is included in Supplementary Figure 5.

Gómez-Morón Á., Requena S., Pertusa C., Lozano-Prieto M., Calzada-Fraile D., Scagnetti C., Sánchez-García I., Calero-García A.A., Izquierdo M, & Martín-Cófreces N.B. (2023). End-binding protein 1 regulates the metabolic fate of CD4+ T lymphoblasts and Jurkat T cells and the organization of the mitochondrial network. Frontiers in Immunology, 14, 1197289.

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Ghost dye red 780 viability dye

Lab products found in correlation

10 protocols using ghost dye red 780 viability dye

Quantifying Immune Cell Profiles in Intracranial Melanoma

Multiparametric flow cytometry analysis of BAL cells

Neutrophil Isolation from Lung Tissue

Flow Cytometric Analysis of Adipose B Cells

Phenotyping Monocyte Subsets by Flow Cytometry

Quantifying Complement-Mediated C3b Deposition

Quantifying Complement-Mediated C3b Deposition

Monocyte Surface Marker Characterization

Characterization of CAR T-cell Tumor Infiltration

Assessing T-cell Viability and Apoptosis

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