Cd8 viogreen

Manufactured by Miltenyi Biotec

Sourced in Germany, United States

CD8-VioGreen is a fluorochrome-conjugated antibody that binds specifically to the CD8 cell surface marker. It can be used for the identification and enumeration of CD8-positive cells in flow cytometric analysis.

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14 protocols using cd8 viogreen

Multiparameter Flow Cytometry of Immune Cells

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The following monoclonal antibodies reactive with human species were used for staining: CD8-VioGreen (BW135/80, cat.: 130-113-726, 1:50), CD4-APC-Vio770 (VIT4, cat.: 130-113-211, 1:50), CD154/CD40L-PE-Vio770 (5C8, cat.: 130-096-793, 1:10), CD20-VioGreen (LT20, cat.: 130-113-379, 1:50), CD14-VioGreen (TÜK4, 130-113-153, 1:50) (all from Miltenyi Biotec, Bergisch Gladbach, Germany);
IL-13-FITC (PVM13-1, cat.: 11-7139-42, 1:20 or 85BRD, cat.: 11-7136-42, 1:20), IL-4-PE (8D4-8, cat.: 12-7049-42, 1:20) (all from Thermo Fisher Scientific); TNFa-Pacific Blue (MAb11, cat.: 502920, 1:100), IFNg-PerCp-Cy5.5 (4S.B3, cat.: 502526, 1:20), CD3-APC (Okt3, cat.: 17-0037-42, 1:50) (all from Biolegend, San Diego, CA, US). Fixation and permeabilization were performed using the Foxp3 Transcription Factor Staining Buffer Set (Thermo Fisher Scientific) according to the manufacturer’s suggestions followed by intracellular cytokine and CD154/CD40-L staining. Fixable viability dye was used in eFluor-506 (cat.: 65-0866-14, 1:1000, Thermo Fisher Scientific).
Cells were acquired using BD FACSCanto II (with Diva software, Heidelberg, Germany) and post-acquisition data analysis was carried out using FlowJo software (TreeStar, Ashland, OR, US).

Ebner F., Morrison E., Bertazzon M., Midha A., Hartmann S., Freund C, & Álvaro-Benito M. (2020). CD4+ Th immunogenicity of the Ascaris spp. secreted products. NPJ Vaccines, 5, 25.

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Multidimensional T-Cell Immunophenotyping

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T‐cell phenotyping was performed on cryopreserved PBMC. Cells were thawed, washed and stained in the dark with fluorescent‐conjugated mononuclear antibodies CD3‐FITC, CD4‐peridinin chlorophyll protein (PerCP), Human Leukocyte Antigen D Related (HLA‐DR)‐allophycocyanin (APC), CD38‐phycoerythrin (PE), CD57‐PE (Becton‐Dickinson, San Diego, CA, USA), CD8‐Viogreen and CD28‐APC (Miltenyi Biotec). Samples were analysed using LSRII Flow cytometer (Becton‐Dickinson). A total of 100,000 events were collected in the lymphocyte gate using morphological parameters (forward and side‐scatter). Data were processed with FACSDiva Software (Becton‐Dickinson) and analysed using Kaluza Analysis Software v.1.2 (Beckman Coulter). Live/Dead Fixable Near‐IR Dead Cell Stain Kit (Life Technologies, Carlsbad, CA, USA) was employed to stain and exclude dead cells.

Dalzini A., Ballin G., Dominguez‐Rodriguez S., Rojo P., Petrara M.R., Foster C., Cotugno N., Ruggiero A., Nastouli E., Klein N., Rinaldi S., Pahwa S., Rossi P., Giaquinto C., Palma P, & De Rossi A. (2021). Size of HIV‐1 reservoir is associated with telomere shortening and immunosenescence in early‐treated European children with perinatally acquired HIV‐1. Journal of the International AIDS Society, 24(11), e25847.

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Multiparameter Flow Cytometry Staining

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Cells were stained with a viability dye (Live/Dead fixable near-IR dead cell stain kit, Invitrogen) and fluorochrome-labelled antibodies for 30 minutes at 4°C. The following antibodies were used: CD3-eFluor450 (OKT3, eBioscience), CD8-VioGreen (BW135/80, Miltenyi), γδTCR-FITC (5A6.E9, Invitrogen), CD161-PE and CD161-APC (191B8, Miltenyi), Vδ2-PerCP-Cy5.5 (B6, BioLegend), CD56-PE-Cy7 (HCD56, BioLegend), Vα7.2-APC and Vα7.2-PerCP-Cy5.5 (3C10, BioLegend), CCR7-FITC (G043H7), CD62L-PE (DREG-56, BioLegend). Data was acquired by a MACSQuant Analyzer 10 (Miltenyi).

Hagel J.P., Bennett K., Buffa F., Klenerman P., Willberg C.B, & Powell K. (2021). Defining T Cell Subsets in Human Tonsils Using ChipCytometry. The Journal of Immunology Author Choice, 206(12), 3073-3082.

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Immune Cell Profiling in Inflammatory Bowel Disease

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Flow cytometry was performed on the dissociated colon cell suspension using a BD LSR Fortessa (BD Bioscience) and analyzed with Diva Software. Viable cells were negative for the staining with Live/Dead Fixable Violet dead cell stain (Lifetechnology). For the evaluation of immune populations, cells were then labeled with the following antibodies: CD45-PerCPVio700, CD11b-FITC, F4/80-APC, CD3-PE, CD8-VioGreen, CD4-VioBrightFITC, and CD25-PEVio770 (Miltenyi Biotec).
T lymphocyte profiling studies were performed by intracellular staining with the inside Stain Kit and the following antibodies: IFN-γ-FITC, IL-2-PE, and TNF-α-APC for the Th1 profile and IL-10-FITC, IL-4-PE, and IL-5-APC for the Th2 profile and Treg were evaluated with Foxp3-Vio667 as recommended by the manufacturer (Miltenyi Biotec).
Appropriate fluorochrome-matched isotype antibodies were used to determine nonspecific background staining. All stainings were performed on 100 µl of PBS without calcium and magnesium and 1% heat-inactivated FCS.
A multiplex bead-based immunoassay was used (Biolegend, LEGENDPlex™ Mix and Match System) for cytokine and Arginase-1 titration from IBD patients’ monocytes.

Rahabi M., Salon M., Bruno-Bonnet C., Prat M., Jacquemin G., Benmoussa K., Alaeddine M., Parny M., Bernad J., Bertrand B., Auffret Y., Robert-Jolimaître P., Alric L., Authier H, & Coste A. (2022). Bioactive fish collagen peptides weaken intestinal inflammation by orienting colonic macrophages phenotype through mannose receptor activation. European Journal of Nutrition, 61(4), 2051-2066.

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Flow Cytometric Analysis of CD8+ T cells

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Flow cytometric analysis of experiments was performed on a Gallios Flow Cytometer (Beckmann Coulter, Brea, CA, USA). Antibodies used for CD8⁺ T cm enrichment were: CD62L-phycoerythrin (PE) (clone: 145/15; Miltenyi, Bergisch-Gladbach, Germany), CD45RO-PE-Vio770 (clone: UCHL1; Miltenyi), and propidium iodide (PI) (Thermo Fisher Scientific, Waltham, MA, USA). For flow cytometric analysis the following antibodies were used: CD3-PE-Vio615 (clone: REA613; Miltenyi), CD8-peridinin chlorophyll protein complex (PerCP)-Vio700 (clone: BW 135/80; Miltenyi), CD8-VioGreen (clone: BW 135/80; Miltenyi), and CD45-V450 (clone: HI30) (BD Biosciences). Live/dead cell discrimination was performed with 7-AAD (Sigma-Aldrich, St. Louis, MO, USA) and Viobility 405/520 Fixable Dye (Miltenyi). Cell doublets were excluded from analysis.

Melzer M.K., Zeitlinger L., Mall S., Steiger K., Schmid R.M., Ebert O., Krackhardt A, & Altomonte J. (2018). Enhanced Safety and Efficacy of Oncolytic VSV Therapy by Combination with T Cell Receptor Transgenic T Cells as Carriers. Molecular Therapy Oncolytics, 12, 26-40.

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Quantifying Cytotoxicity and Delivery

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For cytotoxicity experiments, transiently transfected HEK293T cells were harvested for analysis at 24 hours (SUMO-GrB) or 48 hours (ENLYFQ-GrB) post-transfection. Culture media containing any dislodged cells were collected in centrifuge tubes before adherent cells were trypsinized and subsequently collected into the same tubes. The cells were washed twice with PBS prior to staining with 7-AAD (Life Technologies), DRAQ7 (eBioscience, San Diego, CA), or Annexin V-FITC (BioLegend, San Diego, CA), according to manufacturer’s protocols. For GrB-mCherry delivery experiments, primary human T cells expressing CD19 CAR and GrB-mCherry were co-cultured with 20,000 Raji cells at 3:1 effector:target ratio for 45 min. Co-cultures were then suspended by rigorous pipetting and stained with CD8-VioGreen (clone BW135/80; Miltenyi Biotec, San Diego, CA) and CD19-VioBlue (clone LT19; Miltenyi Biotec) prior to data acquisition on a MACSQuant VYB flow cytometer (Miltenyi Biotec). Compensation and data analysis were performed using FlowJo Data Analysis software (TreeStar, Ashland, OR).

Ho P., Ede C, & Chen Y.Y. (2017). Modularly Constructed Synthetic Granzyme B Molecule Enables Interrogation of Intracellular Proteases for Targeted Cytotoxicity. ACS synthetic biology, 6(8), 1484-1495.

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Flow Cytometry Immunophenotyping Panel

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MACSQuant Analyzer, MACSQuant Analyzer VYB (Miltenyi Biotec), or FACSCanto, LSRII (Becton Dickinson, NJ) was used to analyze cell populations by flow cytometry. The following antibody and protein conjugates were used: CD3-PE, CD3-APC-Vio770, CD4-APC, CD4-VioGreen, CD8-APC-Vio770, CD8-VioGreen, CD14-APC, CD16-PE, CD19-APC, CD20-PerCP-Vio770, CD20-PE-Vio770, CD34-APC, CD34-PE, CD45-VioBlue, CD45-VioGreen, CD45RO-PE-Vio770, CD56-PE, CD62L-VioBlue, CD95-APC, CD45-VioBlue (mouse) (all from Miltenyi Biotec); ErbB2-Fc fusion protein (R&D Systems), anti-human-IgG (Fc gamma-specific) PE (eBioscience). 7AAD staining was used for dead cell exclusion and Violet Cell Trace (Thermo Fisher) for SupT1 cell tracking.

Radek C., Bernadin O., Drechsel K., Cordes N., Pfeifer R., Sträßer P., Mormin M., Gutierrez-Guerrero A., Cosset F.L., Kaiser A.D., Schaser T., Galy A., Verhoeyen E, & Johnston I.C. (2019). Vectofusin-1 Improves Transduction of Primary Human Cells with Diverse Retroviral and Lentiviral Pseudotypes, Enabling Robust, Automated Closed-System Manufacturing. Human Gene Therapy, 30(12), 1477-1493.

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Comprehensive Immune Cell Profiling

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Antibodies/dyes and dilutions used were: viability dye live/dead fixable-violet (L34955, Invitrogen, 1:1250), CD3-eFluor450 (48-0038, eBioscience, 1:100), CD3-PECy7 (25-0038, eBioscience, 1:100), CD4-VioGreen (130-096-900, Miltenyi Biotech, 1:50), CD8-VioGreen (130-098-062, Miltenyi Biotech, 1:50), CD8-V450 (560347, BD, 1:50), CD8-PerCP.Cy5.5/PerCP (301032, Biolegend, 1:100), CD38-APC (555462, BD, 1:50), CD69-FITC (11-0699, eBioscience, 1:40), CD161-APC (130-098-908, Miltenyi Biotech, 1:100), CD161-PE (130-099-193, Miltenyi Biotech, 1:100), IFN-γ-FITC (130-091-641, Miltenyi Biotech, 1:50), IFN-γ-APCCy7 (502529 Biolegend, 1:50), Vα7.2-PE/PeCy7/APC/FITC (351705/351711/351707/351703, Biolegend, 1:50). Granzyme B-APC (MHGB05, Invitrogen), IL-18Ra-APC (17-7183-41, eBioscience, 1:50), TNF-α-PeCy7 (502929, Biolegend, 1:100). All data was acquired on a MACSQuant (Miltenyi Biotech) or a BD FACSVerse (BD) and analyzed on FlowJo (Tree Star Inc.). Gating strategy is shown in Supplementary Fig. 12.

van Wilgenburg B., Scherwitzl I., Hutchinson E.C., Leng T., Kurioka A., Kulicke C., de Lara C., Cole S., Vasanawathana S., Limpitikul W., Malasit P., Young D., Denney L., Moore M.D., Fabris P., Giordani M.T., Oo Y.H., Laidlaw S.M., Dustin L.B., Ho L.P., Thompson F.M., Ramamurthy N., Mongkolsapaya J., Willberg C.B., Screaton G.R, & Klenerman P. (2016). MAIT cells are activated during human viral infections. Nature Communications, 7, 11653.

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CVID Patient PBMC Stimulation and Cytokine Analysis

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PBMCs isolated from CVIDs patients were cultured in 96-well cell plates at 1x10⁶ cells/well concentration in RPMI 1640 culture medium containing 5% AB serum. PBMCs were cultured at 37°C, 5% CO₂, with CytoStim^® (from the SARS-CoV-2 Prot_S PBMC Kit, Miltenyi, Biotech) for a total of six hours. After two hours of incubation, Brefeldin A (SARS-CoV-2 Prot_S PBMC Kit, Miltenyi, Biotech) was added to the cells to inhibit the transport of proteins to the cellular membrane. Cells were fixed, permeabilized, and stained with CD3 APC, CD4 Vio B515, CD8 Vio Green, CD154 (CD40L) APC Vio 770, IFNγ PE, and TNFα PE-Vio770 (SARS-CoV-2 Prot_S PBMC Kit protocol, Miltenyi Biotech) with the addition of CD45RO BUV395(BD Biosciences).
T cells were gated as CD3+ and divided into CD3+CD4+ and CD3+CD8+ T cells. Naïve T cells were gated as CD3+CD4+CD45RO- (or CD8+) and memory T cells were identified as CD3+CD4+CD45RO+ (or CD8+). The CD40L, IFNγ, and TNFα were evaluated on CD4 and CD8 memory T cells. Cells were acquired on a BD FACSymphony A5™ and data were analyzed with FlowJo ver. 10.8.1 (BD Bioscience).

Piano Mortari E., Pulvirenti F., Marcellini V., Terreri S., Salinas A.F., Ferrari S., Di Napoli G., Guadagnolo D., Sculco E., Albano C., Guercio M., Di Cecca S., Milito C., Garzi G., Pesce A.M., Bonanni L., Sinibaldi M., Bordoni V., Di Cecilia S., Accordini S., Castilletti C., Agrati C., Quintarelli C., Zaffina S., Locatelli F., Carsetti R, & Quinti I. (2023). Functional CVIDs phenotype clusters identified by the integration of immune parameters after BNT162b2 boosters. Frontiers in Immunology, 14, 1194225.

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Immunophenotyping of PBMC and T-cells

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Staining of cell surface markers on PBMC and T-cells was performed with CD3-APC/Vio770, CD4-Vioblue, CD8-Viogreen, CD19-FITC, CD56-PE/Vio770, CD16-PE, CD62L-Vioblue, CD45RA-PE, CD45RO-APC, and CCR7-FITC (Miltenyi, San Diego, CA; BD, Franklin Lakes, NJ). All samples were acquired on a MACSQuant Cytometry (Miltenyi) and the data analyzed with Flow Jo (Treestar, Ashland, OR).

McLaughlin L.P., Lang H., Williams E., Wright K.E., Powell A., Cruz C.R., Colberg-Poley A.M., Barese C., Hanley P.J., Bollard C.M, & Keller M.D. (2016). Human Parainfluenza Virus-3 can be Targeted by Rapidly ex vivo Expanded T-Lymphocytes. Cytotherapy, 18(12), 1515-1524.

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