Cd4 viogreen

Manufactured by Miltenyi Biotec

Sourced in United States

The CD4-VioGreen is a fluorochrome-conjugated antibody for the detection and enumeration of CD4-positive cells in flow cytometric analysis. It binds specifically to the CD4 surface antigen expressed on a subset of T lymphocytes.

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Anti-CD4-VioGreen

12 protocols using cd4 viogreen

Quantifying Immune Cell Subsets

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For cell phenotype analysis, monoclonal antibodies were directly added to 100 μL aliquots of heparinized whole blood and incubated at room temperature for 15 min. The antibodies used were: CD3 PECy7, CD14 APC, CD56 PE-Cy5 and CD19 PerCp (Biolegend, San Diego, CA, USA), CD4 Viogreen (Miltenyi Biotec GmBh, Bergisch Gladbach, Germany), CD39 Fitc, CD73 PE, CD25 BV421 and CD127 AlexaFluor 647 (BD Biosciences, San Jose, CA, USA). Red blood cells were lysed and white cells were fixed using BD FACS lysing solution (BD Bioscience). Negative populations were determined using respective anti-human isotype controls. Samples were acquired using a MACSQuant Analyzer 10 flow cytometer (Miltenyi Biotec GmBh) and the data were analyzed using FlowJo software v. 10.0 (TreeStar Inc., Ashland, OR, USA).
We compared the frequencies of four subsets based on CD39 and CD73 expression (CD39⁻CD73⁺, CD39⁺CD73⁺, CD39⁺CD73⁻, and CD39⁻CD73⁻) within each studied population (B cells as CD3⁻CD19⁺, CD4⁺ T cells as CD3⁺CD4⁺, CD8⁺ T cells as CD3⁺CD4⁻, NK cells as CD3⁻CD4⁻CD19⁻CD56⁺, Tregs as CD3⁺CD4⁺CD25⁺⁺CD127⁻, and monocytes as CD14⁺). The percentage of these four subsets was related to their corresponding precursor populations (CD4⁺ T, CD8⁺ T, B, NK, and monocytes). In the case of Treg, the percentage was related to CD4⁺ T cells.

Ortiz M.A., Diaz-Torné C., De Agustin J.J., Estrada P., Reina D., Hernandez M.V., Sang H., Zamora C., Cantó E., Corominas H, & Vidal S. (2023). Altered CD39 and CD73 Expression in Rheumatoid Arthritis: Implications for Disease Activity and Treatment Response. Biomolecules, 14(1), 1.

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Multiparameter Immunophenotyping of Cells

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CD4-VioGreen (130-106-655, Miltenyi Biotec), Anti-CCR10-PE (clone: REA326, 130-104-822, Miltenyi Biotec), CD183 (CXCR3)-PE-Vio770 (clone: REA326, 130-104-822, Miltenyi Biotec), CD194 (CCR4)-APC (clone: REA279, 130-103-813, Miltenyi Biotec), CD196 (CCR6)- PE-Vivo-615 (clone: REA190, 130-107-142, Miltenyi Biotec), Propidium iodide (130-093-233, Miltenyi Biotec), NESTIN rabbit-anti-mouse (ab7659, Abcam), SCA1 rat-anti-mouse (ab25195, Abcam), PDGFRα goat-anti-mouse (AF1062, R&D Systems), Alexa Flour 405 donkey-anti-Rabbit (ab175651, Abcam), Alexa Flour 488 donkey-anti-goat (A-11055, Thermo Fisher Scientific), Alexa Flour 594 donkey-anti-rat (A-21209, Thermo Fisher Scientific), PerCP-CyTM 5.5 CD45.2 conjugated mouse-anti-mouse (552950, BD Biosciences), ICAM1 rabbit-anti-human (ab53013, Abcam), VCAM1 rabbit-anti-human (ab134047, Abcam), IL-1β rabbit-anti-human (ab2105, Abcam), TNF-α rabbit-anti-human (ab9635, Abcam), actin rabbit-anti-human (A2066, Sigma-Aldrich), rabbit anti goat (81–1620, Invitrogen), goat anti rabbit (65–6120,Invitrogen), CD3 rabbit-anti mouse (AD452, Dako), and B220 rat-anti-mouse (MAB1217, R&D Systems).

Dorraji S.E., Hovd A.M., Kanapathippillai P., Bakland G., Eilertsen G.Ø., Figenschau S.L, & Fenton K.A. (2018). Mesenchymal stem cells and T cells in the formation of Tertiary Lymphoid Structures in Lupus Nephritis. Scientific Reports, 8, 7861.

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Flow Cytometry Profiling of T-Cell Subsets

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The following antibodies were used: Vα7.2-PE, CD3-PECy7 (BioLegend, San Diego, CA, USA), CD4-VioGreen, CD161-APC (Miltenyi Biotec, Bergisch Gladbach, Germany), CD4-APCeFluor780, CD8-eFluor450 (eBiosciences, San Diego, CA, USA), CD3-Pacific Orange (Life Technologies, Carlsbad, CA, USA), CD8-PECy7 (BD Biosciences, San Jose, CA, USA). All samples were stained with Live/Dead Fixable Near IR dye (Life Technologies, Carlsbad, CA, USA). Samples were stained as previously described.^{13 (link)} Flow cytometry was performed on a MACSQuant (Miltenyi Biotec, Bergisch Gladbach, Germany), an LSRII (BD Biosciences, San Jose, CA, USA), or for sorting experiments, on a FACSAria (BD Biosciences, San Jose, CA, USA). Samples were gated on lymphocytes/alive/CD3+ or lymphoctyes/alive/CD3+/CD4− (Supplementary Figure 2, http://links.lww.com/MD/A337). Analysis was performed in FlowJo 9.6 (Treestar, Inc., San Carlos, CA, USA).

Ussher J.E., Phalora P., Cosgrove C., Hannaway R.F., Rauch A., Günthard H.F., Goulder P., Phillips R.E., Willberg C.B, & Klenerman P. (2015). Molecular Analyses Define Vα7.2-Jα33+ MAIT Cell Depletion in HIV Infection: A Case–Control Study. Medicine, 94(29), e1134.

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Multiparameter Flow Cytometry of Brain Immune Cells

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Staining of brain immune cell surface antigens was performed as previously described^{47 (link)}. Fc receptors were blocked with 2.4G2 antibody. Cells were incubated with the appropriate combination of conjugated antibodies: CD11b-PercP-Cy5.5, CD45-APC-Cy7, Ly6C-PE-Cy7, Ly6G-pacific blue, CD3-FITC, CD8-APC, major histocompatibility complex class II-Alexa700, CD80-V450, CD86-eFluor605, CD14-APC, TLR4-Alexa488, CD124/IL-R4α-Biotin and streptavidin-PE-Cy7 (BD Biosciences), CD4-Viogreen (Miltenyi Biotec), CCR2-PE (R&D Systems) or isotype control antibodies for 30 minutes. Cells were washed and resuspended in PBS containing 0.5% bovine serum albumin (BSA) for analysis and cell sorting with FACS Aria III (BD Biosciences).

Widmann C., Gandin C., Petit-Paitel A., Lazdunski M, & Heurteaux C. (2018). The Traditional Chinese Medicine MLC901 inhibits inflammation processes after focal cerebral ischemia. Scientific Reports, 8, 18062.

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Multiparametric Flow Cytometry Analysis

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Data were acquired on a MACSQuant® Analyzer 10 or MACSQuant® X (Miltenyi Biotec) and analyzed with FlowLogic™ V.8 (Inivai Technologies) by either frequency or median fluorescence intensity (MFI). Cells were stained in CliniMACS® PBS/EDTA Buffer (Miltenyi Biotec, catalog no.: 200-070-025) with 0.5% BSA at 4°C for 10 min. Antibodies used for flow cytometry: CD137-APC (clone: REA765, Miltenyi Biotec), CD25-PE-Vio770 (clone: REA570, Miltenyi Biotec), CD69-VioBlue (clone: REA824, Miltenyi Biotec), LNGFR-PE (clone: REA844, Miltenyi Biotec), CD4-VioGreen (clone: REA623), CD8-APC-Vio770 (clone: REA734, Miltenyi Biotec), CD3-FITC (clone: REA613, Miltenyi Biotec), CD45-VioBlue (clone: REA747, Miltenyi Biotec), CD279-PE-Vio770 (clone: REA1165, Miltenyi Biotec), CD366-APC (clone: REA635, Miltenyi Biotec), CD223-FITC (clone: REA351, Miltenyi Biotec). Antibodies were used according to the manufacturer’s protocol.

Werchau N., Kotter B., Criado-Moronati E., Gosselink A., Cordes N., Lock D., Lennartz S., Kolbe C., Winter N., Teppert K., Engert F., Webster B., Mittelstaet J., Schaefer D., Mallmann P., Mallmann M.R., Ratiu D., Assenmacher M., Schaser T., von Bergwelt-Baildon M., Abramowski P, & Kaiser A.D. (2022). Combined targeting of soluble latent TGF-ß and a solid tumor-associated antigen with adapter CAR T cells. Oncoimmunology, 11(1), 2140534.

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Immunophenotyping of Brain Immune Cells

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Staining of brain immune cell surface antigens was performed as follows. In brief, Fc receptors were blocked with 2.4G2 antibody. Cells were incubated with the appropriate combination of conjugated antibodies: CD11b-PercP-Cy5.5, CD45-APC-Cy7, Ly6C-PE-Cy7, Ly6G-pacific blue, CD3-FITC, CD8-APC, major histocompatibility complex class II-Alexa700, CD80-V450, CD86-eFluor605, CD14-APC, TLR4-Alexa488, CD124/IL-4Rα-Biotin and streptavidin-PE-Cy7 (BD Biosciences), CD4-Viogreen (Miltenyi Biotec, Paris, France), CCR2-PE (R&D systems, Lille, France) or isotype control antibodies for 30 minutes. Cells were washed and resuspended in PBS containing 0.5% bovine serum albumin for analysis and cell sorting with FACS Aria III (BD Biosciences).

Cazareth J., Guyon A., Heurteaux C., Chabry J, & Petit-Paitel A. (2014). Molecular and cellular neuroinflammatory status of mouse brain after systemic lipopolysaccharide challenge: importance of CCR2/CCL2 signaling. Journal of Neuroinflammation, 11, 132.

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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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Immunophenotyping of CAR T-cell Infusion Products

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For CAR T-cell differentiation, at least 10⁶ cells from infusion product leftovers were used. Cells were incubated with the CD19 CAR detection reagent (Miltenyi Biotec). After wash, the following antibodies were added: biotin-PE, CD3-FITC, CD4-VioGreen, CD8-APC-Vio770, CD45RO-APC (clone REA611, catalog no. 130–115–556), CD62L-Pe-Vio700 (clone 145/15, catalog no. 130–113–621) and CD197-VioBlue (clone REA546, catalog no. 130–117–353; all from Miltenyi Biotec). Data were acquired on BD FACSCanto II (BD Biosciences) or MACSQuant Analyzer MQ10 (Miltenyi Biotec) and analyzed using FlowJo software (RRID:SCR_008520), version 10. Additional information on differentiation status of CAR T cells within infusion products are reported in Supplementary Fig. S5.

Monfrini C., Stella F., Aragona V., Magni M., Ljevar S., Vella C., Fardella E., Chiappella A., Nanetti F., Pennisi M., Dodero A., Guidetti A., Corradini P, & Carniti C. (2022). Phenotypic Composition of Commercial Anti-CD19 CAR T Cells Affects In Vivo Expansion and Disease Response in Patients with Large B-cell Lymphoma. Clinical Cancer Research, 28(15), 3378-3386.

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Multicolor Flow Cytometry Immunophenotyping

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For external staining, cells from cell lines or PBMCs in PBS were incubated with anti-surface antibodies at room temperature (RT) for 20 min. Live/dead staining was performed using LIVE/DEAD® Fixable Near-IR Dead Cell Stain Kit(Invitrogen), at 633 or 635 nm excitation.
For internal staining, cells were fixed with 2% formaldehyde (Sigma Aldrich) in PBS for 10 min and permeabilized with IX permeabilization buffer (eBioscience) in water.
The following antibodies were used: CD3-FITC (BioLegend, Catalog No. 300406, clone UCHT1, Mouse IgG1, k), CD8-PerCP-Cy5.5 (BioLegend, Catalog No. 344710, clone SK1, Mouse IgG1, k), CD38-PerCP-Cy5.5 (BioLegend, Catalog No. 303522, clone HIT2, Mouse IgG1, k), CD56-APC (Biolegend, Catalog No. 318310, clone HCD56, Mouse IgG1, k); CD19-BV421 (BD Bioscience, Catalog No. 562441, clone HIB19, mouse IgG1, k); CD4-VioGreen (Miltenyi Biotec, Catalog No. 130-106-712, clone M-T466, Mouse IgG1, k), CD161-PE (Miltenyi Biotec, Catalog No. 130-092-677, clone 191B8, Mouse IgG2a), IgG2A isotype control (R&D Systems, Catalog No. MAB003, mouse); and 2H7 mAb. When non-conjugated primary antibodies were used, a secondary rat anti-mouse IgG2A-PE (R&D Systems, Catalog No. F0129, clone 344701, IgG1) was used.
FACS analysis was performed on Miltenyi Biotec MACSQuant cytometer and analyzed with FlowJo Version 9.6.2 software (TreeStar).

Llibre A., Garner L., Partridge A., Freeman G.J., Klenerman P, & Willberg C.B. (2016). Expression of lectin-like transcript-1 in human tissues. F1000Research, 5, 2929.

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