PBMCs were isolated via standard density gradient centrifugation and maintained in RPMI 1640 medium supplemented with 10% fetal bovine serum, 2 mm l ‐glutamine, 100 μg mL−1 streptomycin and 100 U mL−1 penicillin (all from Life Technologies, San Diego, CA, USA). Freshly prepared cells for analysis in the absence of in vitro stimulation were either used directly in flow cytometric experiments or placed in culture overnight for concurrent analysis with stimulated cells. All antibody reagents, either purified or pre‐conjugated, were obtained from BD Biosciences (San Diego, CA, USA), except (1) anti‐TRAV10 (Vα24), anti‐TRBV25 (Vβ11) and anti‐CD127 (Beckman Coulter, Brea, CA, USA); (2) anti‐granzyme B (Caltag, Burlingame, CA, USA); and (iii) anti‐CCR7 (R&D Systems, Minneapolis, MN, USA). Multimeric complexes of PBS57‐hCD1d, a ligand analog of αGalCer‐CD1d, were obtained from the National Institutes of Health Tetramer Facility.
Anti ccr7
Manufactured by R&D Systems
Sourced in United States
Anti-CCR7 is a lab equipment product from R&D Systems. It functions as an antibody that specifically binds to the CCR7 protein, which is a chemokine receptor involved in immune cell trafficking and lymphoid organ development.
Automatically generated - may contain errors
Lab products found in correlation
Anti cd127, by Beckman Coulter (2 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (2 mentions)
Anti cd86, by BD (2 mentions)
Anti cd83, by Beckman Coulter (2 mentions)
Anti cxcr3, by BioLegend (2 mentions)
Cxcl9, by Thermo Fisher Scientific (2 mentions)
Cxcl10, by Thermo Fisher Scientific (2 mentions)
Ccl21, by Thermo Fisher Scientific (2 mentions)
Ccl19, by Thermo Fisher Scientific (2 mentions)
Anti cd56, by Beckman Coulter (2 mentions)
Anti hla dr, by BioLegend (2 mentions)
Penicillin, by Thermo Fisher Scientific (1 mentions)
Streptomycin, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
L glutamine, by Thermo Fisher Scientific (1 mentions)
Live dead fixable yellow dead cell stain, by Thermo Fisher Scientific (1 mentions)
Anti ifn γ, by BD (1 mentions)
Anti cd8, by BD (1 mentions)
Anti cd69, by BioLegend (1 mentions)
Cd8 percp cy5, by BD (1 mentions)
9 protocols using anti ccr7
1
PBMC Isolation and Staining Protocol
2
Multiparametric Phenotyping of T Cell Subsets
3
Flow Cytometry Analysis of Immune Markers
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Flow cytometry was done using the following monoclonal antibodies (mAb) and appropriate isotype controls: anti-HLA class I (W6/32), anti-HLA DR/DP (Q5/13), anti-HLA DR, anti-CD70, anti-CD80 (all BD Biosciences), anti-CD14, anti-CD83 (both Beckman Coulter), anti-CD86 (BD PharMingen), and anti-CCR7 (R&D systems). For intracellular staining of the TAA NKI/beteb (IgG2b; purified antibody) against gp100, and T311 (IgG2a; Cell Marque Corp., Rocklin, CA) against tyrosinase were used. Cells were also stained intracellular with anti-CD40L (Beckman Coulter). For intracellular staining, cells were fixed for 4′ on ice in 4% (w/v) paraformaldehyde (Merck) in PBS, permeabilized in PBS/2%BSA/0.02% azide/0.5% saponin (Sigma-Aldrich) (PBA/saponin), and stained with mAb diluted in PBA/saponin/2%HS, followed by staining with Alexa488-labeled goat-anti-mouse (BD PharMingen). Flow cytometry was performed with FACSCalibur™ flow cytometer equipped with CellQuest software (BD Biosciences).
4
Transwell Migration of T Cells
5
Investigating Protein Interactions and Signaling Pathways
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Cells were lysed in the RIPA buffer (Beyotime) containing 1% Protease/Phosphatase Inhibitor Cocktail (MCE) and PMSF. The BCA Protein Assay Kit (Epizyme) was applied to measure protein concentrations. Western blot analysis was carried out as previously described.22 For coimmunoprecipitation assays, MIA‐PaCa‐2 and SW1990 cells were transfected with the indicated plasmids according to manual of Lipofectamine 3000’s manufacturer (Sigma‐Aldrich). Immunoprecipitation was undertaken as described previously.22 , 23 In this study, anti‐PD‐L1 (ab228415) was purchased from Abcam. Anti‐phospho‐AKT (Ser473)(9271), anti‐AKT (9272), anti‐phospho‐GSK‐3β (ser9)(9323), anti‐GSK‐3β (12456), anti‐GAPDH (5174), and anti‐β‐actin (4970) were purchased from Cell Signaling Technology. Anti‐Ccl21 (AF457‐SP) and anti‐CCR7 (150503) were purchased from R&D Systems.
6
Comprehensive Multiparametric Flow Cytometry Analysis
7
Phenotypic Analysis of Virus-Specific CD8+ T Cells
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Mononuclear cells from blood, spleen and tonsils were stained with fluorochrome-conjugated antibodies (mAbs) specific for cell surface proteins. The following mAbs were used for identification of CD8+ T cells and the determination of their phenotype; anti-CD3, anti-CD8 (Biolegend), anti-CCR7 (R&D Systems), anti-CD45RA (BD Biosciences), anti-CD69, anti-CD25, anti-CD137, anti-HLA-DR, anti-CD103, anti-KLRG-1 and anti-CD11a (all obtained from Biolegend). Fluorochrome-conjugated HLA class I dextramers (Immudex) were used to identify virus-specific CD8+ T cells. EBV-specific CD8+ T cells were identified with dextramers specific for the following viral epitopes; GLCTLVAML (derived from EBV-lytic protein BMLF1), CLGGLLTMV (derived from EBV-latent protein LMP2), RAKFKQLL (derived from EBV-lytic protein BZLF1) and FLRGRAYGL (derived from EBV-latent protein EBNA3A). CMV-specific CD8+ T cells were identified with dextramers specific for the following viral epitopes; NLVPMVATV, TPRVTGGGAM, RPHERNGFTV (all derived from pp65 protein), VLEETSVML, ELRRKMMYM, ELKRKMMYM (all derived from IE-1 protein) and VTEHDTLLY (derived from pp50 protein). Stained cells were analyzed on either FACSCanto II or LSRFortessa flow cytometer (BD Biosciences) and the data processed using FlowJo software (Treestar, Ashland, USA).
8
Comprehensive Lymphocyte Subset Analysis
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The leukapheresis products were suspended in RPMI 1640 medium (Life Technologies, Carlsbad, CA, USA) supplemented with 10% fetal calf serum and were analyzed using flow cytometry. Eight-colour analyses were performed for the identification of the lymphocyte subsets with the following monoclonal antibodies: CD2, CD3, CD4, CD5, CD7, CD16, CD25, CD56, CD45, CD45RA, CD45RO, CD9, CD127, CCR7, and HLA-DR antigens. All antibodies were purchased from BD Biosciences (San José, CA, USA) except for anti-CD8, anti-CD56, and anti-CD127 which were purchased from Beckman Coulter (Brea, CA, USA); anti-CCR7 was purchased from R&D systems (MN, USA) and anti HLA-DR from Biolegend (San Diego, CA, USA). The Blood Dendritic Cell Enumeration Kit (Miltenyi Biotech GmbH, Bergish Gladbach, Germany) was used according to the supplier's protocol to determine plasmacytoid dendritic cells, type 1 and type 2 myeloid dendritic cells. Flow cytometry analysis was performed on the LSR II instrument (BD Biosciences). Data analysis was performed using the Flow-Jo software (Tree Star, Ashland, OR, USA).
9
Transwell Assay for T Cell Migration
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Migration of T cells was assessed by using Transwell permeable supports with 5-μm polycarbonate membrane (Costar, 3387). To determine cell migration in response to soluble factors, the bottom chamber was loaded with 0.1% FBS, 200 ng/mL CCL21 (PeproTech, 250–13), 200 ng/mL CCL19 (PeproTech, 250–27B), 150 ng/mL CXCL9 (PeproTech 250–18), or 50 ng/mL CXCL10 (PeproTech, 250–16) in T-cell medium. T cells were incubated for 2 (naïve) or 3 hours (activated) at 37°C and migrated cells in the bottom chamber were collected and counted by flow cytometry using Precision Count Beads. According to the experimental requirements, activated T cells were pretreated for 1 hour with 10 μg/mL anti-CCR7 (R&D Systems, 4B12), 250 μg/mL anti-CXCR3 (BioLegend, CXCR3–173), 100 μmol/L Rac1 inhibitor NSC23766, or the appropriate isotype, or vehicle control.
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