We pre-incubated 1 × 106–3 × 106 cells with APC-conjugated VY9, or NP8 tetramer at 37°C, 5% CO2 for one hour. (The tetramers were produced by the NIH Tetramer Core Facility at Emory University, Atlanta, GA.) Next, we added the antibodies recognizing the surface phenotypic markers. We used antibodies from BD Biosciences: CCR7 (clone 150503, R&D Systems) FITC, CD3 (clone SP34-2) PE-CF594, CD8 (clone RPA-T8) BV711, CD20 (clone 2H7) Alexa700, CD28 (clone CD28.2,) PE, CD45 (clone D058-1283) BV786; from BioLegend: PD-1 (clone EH12.2H7) BV605; from Beckman Coulter: NKG2a (clone Z199) PE-Cy7, and Near-Infrared Live/Dead Discriminator from Life Technologies. We incubated the cells for 15 min at room temperature, removed any unbound reagents with two washing steps then fixed them with 2% PFA. We acquired the data on a special-order BD LSR II (BD Biosciences, San Jose, CA) equipped with a 50 mW 405 nm violet, a 100 mW 488 nm blue, and a 50 mW 640 nm red laser, 16 detectors, and FACSDiva software version 8.0.1. We collected up to 250,000 events in the lymphocyte gate defined by the forward and side scatter parameters. We analyzed the data using FlowJo version 10.4.2.
Ccr7 clone 150503
Manufactured by R&D Systems
CCR7 (clone 150503) is a mouse monoclonal antibody that recognizes the chemokine receptor CCR7. CCR7 is a G protein-coupled receptor that plays a role in lymphocyte trafficking and homing to lymphoid organs.
Automatically generated - may contain errors
Lab products found in correlation
Cd3 clone sp34 2 pe cf594, by BioLegend (2 mentions)
Cd8 bv711 clone rpa t8, by BioLegend (2 mentions)
Cd20 clone 2h7 alexa700, by BioLegend (2 mentions)
Pd 1 clone eh12.2h7 bv605, by Beckman Coulter (2 mentions)
Nkg2a clone z199 pe cy7, by Thermo Fisher Scientific (2 mentions)
Near infrared live dead discriminator, by Thermo Fisher Scientific (2 mentions)
Fluorescein isothiocyanate (fitc), by R&D Systems (2 mentions)
Lsr 2, by BD (2 mentions)
2 protocols using ccr7 clone 150503
1
Multiparameter Flow Cytometry Phenotyping
2
Multiparametric Flow Cytometry of T-cell Subsets
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We pre‐incubated 1 × 106–3 × 106 cells with APC‐conjugated VY9, or NP8 tetramer at 37°C, 5% CO2 for one hour. (The tetramers were produced by the NIH Tetramer Core Facility at Emory University, Atlanta, GA.) Next, we added the antibodies recognizing the surface phenotypic markers. We used antibodies from BD Biosciences: CCR7 (clone 150503, R&D Systems) FITC, CD3 (clone SP34‐2) PE‐CF594, CD8 (clone RPA‐T8) BV711, CD20 (clone 2H7) Alexa700, CD28 (clone CD28.2,) PE, CD45 (clone D058‐1283) BV786; from BioLegend: PD‐1 (clone EH12.2H7) BV605; from Beckman Coulter: NKG2a (clone Z199) PE‐Cy7, and Near‐Infrared Live/Dead Discriminator from Life Technologies. We incubated the cells for 15 min at room temperature, removed any unbound reagents with two washing steps then fixed them with 2% PFA. We acquired the data on a special‐order BD LSR II (BD Biosciences, San Jose, CA) equipped with a 50 mW 405 nm violet, a 100 mW 488 nm blue, and a 50 mW 640 nm red laser, 16 detectors, and FACSDiva software version 8.0.1. We collected up to 250,000 events in the lymphocyte gate defined by the forward and side scatter parameters. We analyzed the data using FlowJo version 10.4.2.
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