Ccr7 pe dazzle594

Manufactured by BioLegend

CCR7 PE-Dazzle594 is a fluorescently-labeled antibody that specifically binds to the CCR7 protein. It can be used for the identification and analysis of CCR7-expressing cells in flow cytometry applications.

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CCR7-PE (26 citations)

3 protocols using ccr7 pe dazzle594

T Cell Phenotyping and Flow Cytometry

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For protein expression analysis following proliferation, CD8 or CD4 T cells were isolated and stimulated as described above. On day 4 of the proliferation assay cells were collected and stained with the following antibodies for surface protein expression: CD3 APC-Cy7 (Biolegend), CD4 AF-700 (Biolegend), CD8 PE-Cy7 (Biolegend), CD45RA BV-605 (Biolegend), CCR7 AF-488 (Biolegend) or CCR7 PE-Dazzle594 (Biolegend), and CD19 PerCP-Cy5.5 (Biolegend). Dead cells were excluded from the analysis by using Zombie-UV (Biolegend). Doublets and double positive CD4/CD8 cells were removed through sequential gating. Flow cytometry acquisition was done using the BD LSRII with BD FACSDiva and Cyteck Aurora. Data was analyzed by FlowJo 10.1r7 and GraphPad Prism 9.

Lerrer S., Tocheva A.S., Bukhari S., Adam K, & Mor A. (2021). PD-1-stimulated T cell subsets are transcriptionally and functionally distinct. iScience, 24(9), 103020.

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Profiling Immune Cell Subsets Post-Vaccination

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On days 0, 1, and 14 after the first immunization, the cellular composition of the PBMCs was analyzed using flow cytometry. Freshly isolated PBMCs were stained with Live/Dead Fixable Blue Dye (Life Technologies, cat# L-23105, 1:40 dilution) and FcR blocking reagent (Miltenyi Biotec, cat# 130-059-901, 1:20 dilution) followed by a panel of antibodies: CD40 FITC (5C3, Biolegend, cat# 334306, 1:20 dilution), NKG2A PE (Z199, Beckman Coulter, cat# IM3291U, 1:40 dilution), CD80 BV421 (L307.4, BD, cat# 564160, 1:40 dilution), CCR7 PE-Dazzle594 (G043H7, Biolegend, cat# 353236, 1:50 dilution), CD123 Per-CP-Cy5.5 (7G3, BD, cat# 558714, 1:80 dilution), CD3 APC-Cy7 (SP34-2, BD, cat# 557757, 1:80 dilution), CD66 APC (TET2, Miltenyi Biotec, cat# 130-118-539, 1:80 dilution), CD70 BV786 (Ki-24, BD, cat# 565338, 1:80 dilution), HLA-DR BV650 (L243, Biolegend, cat# 307650, 1:80 dilution), CD11c PE-Cy7 (3.9, Biolegend, cat# 301608, 1:160 dilution), CD16 AF700 (38 G, BD, cat# 560713, 1:160 dilution), CD20 BV605 (2H7, Biolegend, cat# 302334, 1:160 dilution) and CD14 BV510 (M5E2, Biolegend, cat# 301842, 1:160 dilution). After washing with PBS, samples were fixed using 1% paraformaldehyde (PFA) and acquired on a BD LSRFortessa cell analyzer. The data were analyzed using FlowJo software v.10.7.1 (FlowJo).

Lenart K., Arcoverde Cerveira R., Hellgren F., Ols S., Sheward D.J., Kim C., Cagigi A., Gagne M., Davis B., Germosen D., Roy V., Alter G., Letscher H., Van Wassenhove J., Gros W., Gallouët A.S., Le Grand R., Kleanthous H., Guebre-Xabier M., Murrell B., Patel N., Glenn G., Smith G, & Loré K. (2024). Three immunizations with Novavax’s protein vaccines increase antibody breadth and provide durable protection from SARS-CoV-2. NPJ Vaccines, 9, 17.

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Analysis of CAR T-cell Phenotype

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Standard flow cytometry procedures were used as previously described.¹⁷ (link) Cell-surface CAR expression was detected with a non-commercial allophycocyanin (APC)-labeled antibody that binds the linker of the Hu19 scFv.¹⁷ (link) Antibodies were purchased from BD Biosciences (San Jose, CA, USA): CD3 APC-Cy7 (Clone UCHT1), CD4 brilliant violet 510 (Clone RPA-T4), CD8 R-phycoerythrin (PE)-Cy7/eFluor450 (Clone RPA-T8). The following antibodies were purchased from Biolegend: CD45RA fluorescein isothiocyanate (Clone HI100), CCR7 PE-Dazzle 594 (Clone G043H7), and CD95 PE (Clone DX2). Dead cells were excluded by using 7-amino-actinomycin D (7-AAD; BD Biosciences).
Flow cytometry was performed with a LSRFortessa (BD Biosciences) or FACSymphony A5 (BD Biosciences) cytometers. Flow cytometry analysis was performed using FlowJo (Tree Star, Inc., Ashland, OR, USA).

Amatya C., Weissler K.A., Fellowes V., Lam N., Cutmore L.C., Natrakul D.A., Highfill S.L, & Kochenderfer J.N. (2024). Optimization of anti-CD19 CAR T cell production for treatment of patients with chronic lymphocytic leukemia. Molecular Therapy. Methods & Clinical Development, 32(1), 101212.

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