Anti cd80 apc

Manufactured by Miltenyi Biotec

Sourced in Germany

Anti-CD80-APC is a fluorescent-labeled antibody that binds to the CD80 surface protein. CD80 is a co-stimulatory molecule expressed on antigen-presenting cells and plays a role in T cell activation. The APC (Allophycocyanin) fluorescent dye allows for the detection and analysis of CD80-positive cells using flow cytometry.

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4 protocols using anti cd80 apc

Flow Cytometric Characterization of nDCs

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Purity and phenotype of nDCs after immunomagnetic isolation were determined by flow cytometry with a FACSVerse® (BD biosciences, San Jose, CA) or MACS Quant® (Miltenyi Biotec). For this purpose, the following primary monoclonal antibodies and the appropriate isotype or fluorescence minus one controls were used: anti-CD1c-Viobright-FITC, anti-BDCA2-PE, anti-CD123-APC, anti-CD20-PE-Vio770, anti-CD45-APC-Vio770, anti-CD14-Viogreen, anti-FcεRI-BioBlue, anti-CD14-FITC, anti-CD15-PE, anti-CD56-APC, anti-CD3-VioBlue, anti-HLA-ABC-APC, anti-HLA-DR/DP/DQ-APC, anti-CCR7-APC, anti-CD80-APC, anti-CD83-APC, and anti-CD86-APC (all Miltenyi Biotec). Details are depicted in Supplementary Table 2. The purity of the nDC product was defined as the percentage of nDCs (sum of CD123⁺BDCA2⁺ pDC plus CD1c⁺CD20^- cDC2) of all viable cells in the nDC product. After 6 h of protamine/mRNA stimulation, cytokine production of nDCs was measured in the supernatant by cytometric bead array according to the manufacturer’s instruction (Miltenyi Biotec).

Bol K.F., Schreibelt G., Bloemendal M., van Willigen W.W., Hins-de Bree S., de Goede A.L., de Boer A.J., Bos K.J., Duiveman-de Boer T., Olde Nordkamp M.A., van Oorschot T.G., Popelier C.J., Pots J.M., Scharenborg N.M., van de Rakt M.W., de Ruiter V., van Meeteren W.S., van Rossum M.M., Croockewit S.J., Koeneman B.J., Creemers J.H., Wortel I.M., Angerer C., Brüning M., Petry K., Dzionek A., van der Veldt A.A., van Grünhagen D.J., Werner J.E., Bonenkamp J.J., Haanen J.B., Boers-Sonderen M.J., Koornstra R.H., Boomsma M.F., Aarntzen E.H., Gotthardt M., Nagarajah J., de Witte T.J., Figdor C.G., de Wilt J.H., Textor J., de Groot J.W., Gerritsen W.R, & de Vries I.J. (2024). Adjuvant dendritic cell therapy in stage IIIB/C melanoma: the MIND-DC randomized phase III trial. Nature Communications, 15, 1632.

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Immune Cell Surface Marker Analysis

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After blocking the unspecific binding of Fc-receptor for 10 min on ice in 10 µL of a solution containing 5 mg/ml human IgG (IgG1 66.6%, IgG2 28.5%, IgG3 2.7%, IgG4 2.2%; Flebogamma, Grifols, Frankfurt, Germany), cells were washed in PBS/BSA (DRFZ, Berlin, Germany). Antibody staining was performed for 10 min at 4°C for the detection of surface marker expression using anti-CD14-FITC and anti-CD11b-PE (both from DRFZ, Berlin, Germany), anti-CD16-PE-Vio770, anti-CD80-APC and anti-HLA-DR-VioBlue (all from Miltenyi Biotech, Bergisch Gladbach, Germany; see also Table 1). Apoptosis and necrosis of monocytes were quantified by Annexin V (Biolegend, San Diego, USA) and 7-AAD staining (BD Biosciences, San Jose, USA) (gating strategy provided in Figure S1, reagent concentration provided in Tables 1, 2) according to the manufacturer’s instructions. Data were acquired using a BD FACSCanto™ II (BD Biosciences, San Jose, USA) and processed by FlowJo v7.6.5 (BD Biosciences, San Jose, USA).

Krauss P.L., Pfeiffenberger M., Damerau A., Buttgereit T., Chen Y., Gaber T, & Buttgereit F. (2021). Production of IL-6 and Phagocytosis Are the Most Resilient Immune Functions in Metabolically Compromised Human Monocytes. Frontiers in Immunology, 12, 730672.

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Phenotypic and Functional Analysis of cDC2s and pDCs

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Purity and phenotype of cDC2s and pDCs after immunomagnetic isolation were determined by flow cytometry with a FACSVerse® (BD biosciences) or MACS Quant® (Miltenyi Biotec). Purity was analyzed directly after CliniMACS Prodigy isolation. For this purpose, the following primary monoclonal antibodies (mAbs) and appropriate fluorescence minus one controls were used: anti-CD1c-Viobright FITC, anti-BDCA2-PE, anti-CD123-APC, anti-CD20-PE-Vio770, anti-CD45-APC-Vio770, anti-CD14-Viogreen, anti-FcεRI-BioBlue, anti-CD14-FITC, anti-CD15-PE, anti-CD56-APC and anti-CD3-BioBlue. The phenotype of cDC2s and pDCs after 3 hours of pR stimulation was analyzed using the following mAbs and appropriate isotype controls: anti-HLA-ABC-APC, anti-HLA-DR/DP/DQ-APC, anti-CCR7-APC, anti-CD80-APC, anti-CD83-APC and anti-CD86-APC (all Miltenyi Biotec). After 6 hours of pR stimulation cytokine production of cDC2s and pDCs was measured in the supernatant by cytometric bead array according to the manufacturer’s instruction (Miltenyi Biotec).

Bloemendal M., Bol K.F., Boudewijns S., Gorris M.A., de Wilt J.H., Croockewit S.A., van Rossum M.M., de Goede A.L., Petry K., Koornstra R.H., Figdor C., Gerritsen W.R., Schreibelt G, & de Vries I.J. (2021). Immunological responses to adjuvant vaccination with combined CD1c+ myeloid and plasmacytoid dendritic cells in stage III melanoma patients. Oncoimmunology, 11(1), 2015113.

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Phenotypic Characterization of Isolated mDCs and pDCs

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Purity and phenotype of mDCs and pDCs after CliniMACS isolation were determined by flow cytometry with a FACSVerse (BD Biosciences, San Jose, CA, USA) or MACS Quant (Miltenyi Biotec). The following primary monoclonal antibodies and the appropriate isotype or fluorescence minus one controls were used: anti-CD1c-Viobright FITC, anti-BDCA-2-PE, anti-CD20-PE-Vio770, anti-CD123-APC, anti-CD45-APC-Vio770, anti-CD14-VioGreen, anti-FcεRI-VioBlue, anti-CD14-FITC, anti-CD15-PE, anti-CD56-APC, anti-CD3-BioBlue, anti-HLA-ABC-APC, anti-HLA-DR,DP,DQ-APC, anti-CCR7-APC, anti-CD80-APC, anti-CD83-APC and anti-CD86-APC (all Miltenyi Biotec).

Westdorp H., Creemers J.H., van Oort I.M., Schreibelt G., Gorris M.A., Mehra N., Simons M., de Goede A.L., van Rossum M.M., Croockewit A.J., Figdor C.G., Witjes J.A., Aarntzen E.H., Mus R.D., Brüning M., Petry K., Gotthardt M., Barentsz J.O., de Vries I.J, & Gerritsen W.R. (2019). Blood-derived dendritic cell vaccinations induce immune responses that correlate with clinical outcome in patients with chemo-naive castration-resistant prostate cancer. Journal for Immunotherapy of Cancer, 7, 302.

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