Surface phenotypes were determined using an Epics XL MCL (Beckman Coulter, Brea, CA, USA). The following monoclonal antibodies (mAbs) were purchased from Beckman Coulter: anti‐CD3, anti‐CD4, anti‐CD8, anti‐Vγ9TCR, anti‐CD14, anti‐CD25, anti‐CD45, anti‐CD54, anti‐CD56, anti‐HLA‐DR, anti‐CD40, anti‐CD80, anti‐CD86, anti‐CD11c, anti‐CD36, mouse immunoglobulin (Ig)G1, mouse IgG2 and mouse IgG2b mAbs. Anti‐HLA‐class 1 and anti‐CCR7 mAbs were purchased from Beckton Dickinson (San Jose, CA, USA) and R&D Systems (Minneapolis, MN, USA), respectively. Anti‐TCR Vα24TCR and anti‐TCR Vβ11 mAbs were purchased from Beckman Coulter (Villepinte, France). Anti‐human CD273 (PD‐L2) and CD274 (PD‐L1) mAbs were purchased form eBioscience (San Diego, CA, USA). Anti‐human CD152 (CTLA‐4) and CD279 (PD‐1) mAbs were purchased from BioLegend (San Diego, CA, USA). Anti‐FoxP3 mAb for intracellular staining was purchased from BD Biosciences (Tokyo, Japan). All mAbs were conjugated with fluorescein isothiocyanate (FITC), phycoerythrin (PE), antigen‐presenting cells (APC), extracellular domain (ECD), proprotein convertase (PC)5 or PC7. Z was purchased from Novartis Pharmaceuticals (Basel, Switzerland) and G from Funakoshi Co. Ltd (Tokyo, Japan).
Anti cd8
Manufactured by Beckman Coulter
Sourced in United States
The Anti-CD8 is a laboratory reagent used for the identification and enumeration of CD8-positive cells in human peripheral blood samples. It is designed to be used with flow cytometry instrumentation for immunophenotyping applications.
Automatically generated - may contain errors
Lab products found in correlation
Anti cd4, by Beckman Coulter (3 mentions)
Anti cd56, by Beckman Coulter (2 mentions)
Anti hla dr, by Beckman Coulter (2 mentions)
Anti cd45, by Beckman Coulter (2 mentions)
Anti cd3, by Beckman Coulter (1 mentions)
Anti tcr vβ11, by Beckman Coulter (1 mentions)
Epics xl mcl, by Beckman Coulter (1 mentions)
Anti cd80, by Beckman Coulter (1 mentions)
Anti cd86, by Beckman Coulter (1 mentions)
Anti cd127 pe, by Beckman Coulter (1 mentions)
Anti cd45ro, by Beckman Coulter (1 mentions)
Anti cd4 pc7, by Beckman Coulter (1 mentions)
Fc500, by Beckman Coulter (1 mentions)
Anti cd25 pc5, by Beckman Coulter (1 mentions)
Anti cd3, by BD (1 mentions)
Anti cd56, by BD (1 mentions)
Anti cd19, by BD (1 mentions)
Navios, by Beckman Coulter (1 mentions)
Anti cd45ra, by Beckman Coulter (1 mentions)
Lsrii flow cytometer, by BD (1 mentions)
6 protocols using anti cd8
1
Multiparametric Flow Cytometry Analysis
2
Immunophenotyping of T-cell Subsets
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Immunophenotyping was performed by flow cytometric analysis (five-colour flow cytometer FC-500; Beckman Coulter with CXP software).
For the staining of T-lymphocyte differentiation stages the following antibodies were used: R-Phycoerythrin-Cyanine 7 (PC7)-conjugated anti-CD4, anti-CD8, Phycoerythrin-Texas red X (ECD)-conjugated anti-CD8, anti-CD45RO; PC5-conjugated anti-CD27, and anti-CD25 (all from Beckman Coulter). For the staining of Tregs we used anti-CD4 PC7, anti-CD127 PE, and anti-CD25 PC5 (Beckman Coulter).
T lymphocytes, both CD4 and CD8, were divided into naïve CD45RO-CD27+, early differentiated (ED) CD45RO+CD27+, late differentiated (LD) CD45RO+CD27-, and fully differentiated effector (FD) CD45RO-CD27- memory T cells. In CD8+ cells a unique CD45RO-CD27dim intermediate population was determined as well [22 (link)] (Fig. 1 ). Treg cells were defined as CD4+CD25highCD127low [24 (link)] in accordance with recent studies that demonstrated that the description of Tregs as a CD4+CD25highCD127low phenotype correlates well with FoxP3 expression [25 (link), 26 (link)].
For the staining of T-lymphocyte differentiation stages the following antibodies were used: R-Phycoerythrin-Cyanine 7 (PC7)-conjugated anti-CD4, anti-CD8, Phycoerythrin-Texas red X (ECD)-conjugated anti-CD8, anti-CD45RO; PC5-conjugated anti-CD27, and anti-CD25 (all from Beckman Coulter). For the staining of Tregs we used anti-CD4 PC7, anti-CD127 PE, and anti-CD25 PC5 (Beckman Coulter).
T lymphocytes, both CD4 and CD8, were divided into naïve CD45RO-CD27+, early differentiated (ED) CD45RO+CD27+, late differentiated (LD) CD45RO+CD27-, and fully differentiated effector (FD) CD45RO-CD27- memory T cells. In CD8+ cells a unique CD45RO-CD27dim intermediate population was determined as well [22 (link)] (
3
Multiparameter Immunophenotyping of PBMCs
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4
CD8+ T Cell Activation Monitoring
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Activation of CD8+ T cells was assessed by flow cytometry analysis on EDTA-treated fresh whole blood using FC 500 apparatus (Beckman Coulter, Miami, Florida). T cell activation was explored at day 0, week 4 and week 12 after initiation of pegIFNα-ribavirin therapy. The expression of CD38bright on CD8+ T cells was analyzed as previously described using a two-colour staining with anti-CD8 and anti-CD38 conjugated to fluorescein isothiocyanate (FITC) and phycoerythrin (PE), respectively (Beckman Coulter).26 (link) The positive threshold for CD38bright analysis was established using the CellQuant CD38/CD8 kit for quantitation of CD38 cell surface expression (BioCytex, Marseille, France) and was defined as 8,500 CD38 binding sites/cells. The CD8+/CD38bright values were expressed as the percentage of CD38bright cells from the CD8+ T cell populations.
5
Evaluating Tumor Immune Response to Oncolytic Virus and BiTE
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Raji lymphoma cells were implanted intradermally into the right flanks of NSG mice (5 × 106 cells). Mice bearing Raji subcutaneous tumor were treated intratumorally with PBS, OVV, OVV-CD19BiTE (2 × 107 pfu/tumor), or blinatumomab (0.25 mg/kg). Twenty-four hours later, 1 × 107 preactivated T cells were intravenously injected into treated mice. Tumors were harvested 3 days after the injection with forceps and surgical scissors and were weighed. They were then minced before incubation with Liberase (1.67 Wünsch U/mL) and DNase (0.2 mg/mL) in serum-free RPMI for 30 min at 37 °C. Cell suspensions were generated by mashing through a 70 μm nylon filter and then washed with complete RPMI. For tumor-infiltrating T-cells analysis, single-cell suspensions were generated and processed for surface labeling with anti-CD3, anti-CD45, anti-CD4, anti-CD8, anti-HLA-DR, anti-CD45RA, anti-CCR7, anti-CXCR3, and anti-CCR6 antibodies (Beckman). Live cells were distinguished from dead cells by using fixable dye eFluor506 (eBioscience). All flow data were acquired using either an LSRII flow cytometer (BD Biosciences). Data were analyzed with FlowJo software (Treestar).
6
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).
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