Anti ccr7 apc

Manufactured by Miltenyi Biotec

Anti-CCR7-APC is a fluorescently labeled antibody that binds to the CCR7 surface receptor. CCR7 is involved in the trafficking and homing of lymphocytes. The Anti-CCR7-APC antibody can be used to identify and quantify CCR7-expressing cells in flow cytometry applications.

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4 protocols using anti ccr7 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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Characterization of moDC and mDC Responses

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6×10⁵ mDCs or moDCs were cultured for 18 h at 37°C in 200 µL CellGro GMP DC medium (CellGenix, Cat. No.: 20801-0500) supplemented with 50 µg/mL AfuLy, 1-5 µg/mL Aspergillus proteins CcpA or Shm2, 1 µg/mL LPS (Sigma, positive control), or plain medium (negative control). Thereafter, DCs were stained with anti-CD1a-APC (moDCs) or anti-CD1c-APC (mDCs), anti-CD14-FITC, anti-CD80-APC, anti-CD83-PE, anti-CD86-FITC, anti-CD40-FITC, anti-HLA-ABC-PE, anti-HLA-DR-PE (BD), and anti-CCR7-APC (Miltenyi Biotec) in three different panels. Samples were analyzed using a FACS Calibur cytometer (BD), Cell Quest Pro software (BD), and Flow Jo software (version 10.6.2.). Dead cells were excluded from analysis by light scatter (FSC/SSC) properties.

Page L., Wallstabe J., Lother J., Bauser M., Kniemeyer O., Strobel L., Voltersen V., Teutschbein J., Hortschansky P., Morton C.O., Brakhage A.A., Topp M., Einsele H., Wurster S, & Loeffler J. (2021). CcpA- and Shm2-Pulsed Myeloid Dendritic Cells Induce T-Cell Activation and Enhance the Neutrophilic Oxidative Burst Response to Aspergillus fumigatus. Frontiers in Immunology, 12, 659752.

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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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