Cells were labeled with APC-Hy7-conjugated anti-CD3 (BD Biosciences), PE-Cy7-conjugated anti-CD8 (BD Biosciences), or PE-conjugated anti-mouse TCRβ (eBioscience). E629-38/HLA-A*02:01 and E711-19 monomers were provided by the NIH Tetramer Core Facility. Tetramers were assembled and labeled following NIH Tetramer Core Facility protocols. Data were acquired with a BD FACSCanto II flow cytometer (BD Biosciences) and analyzed with FlowJo software (FlowJo, LLC).
Pe cy7 conjugated anti cd8
Manufactured by BD
Sourced in United States
PE-Cy7-conjugated anti-CD8 is a fluorescently labeled antibody that binds to the CD8 protein. It can be used to identify and quantify CD8-positive cells in flow cytometry applications.
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Lab products found in correlation
Apc conjugated anti cd3, by BD (2 mentions)
Pe conjugated anti foxp3, by BD (2 mentions)
Fitc conjugated anti cd4, by BD (2 mentions)
Percpcy5.5 conjugated anti cd19, by BD (2 mentions)
Apc cy7 conjugated anti cd4, by BD (2 mentions)
Pe conjugated anti ly6g, by BD (2 mentions)
Apc conjugated anti cd25, by BD (2 mentions)
Fitc conjugated anti cd49b, by BD (2 mentions)
Fitc conjugated anti ly6c, by BD (2 mentions)
Pe cy7 conjugated anti cd3, by BD (2 mentions)
Facsdiva, by BD (2 mentions)
Mouse foxp3 buffer set, by BD (2 mentions)
Infinicyt, by Cytognos (2 mentions)
Facscanto 2 flow cytometer, by BD (2 mentions)
Software, by FlowJo (1 mentions)
Facscanto 2, by BD (1 mentions)
Gm csf, by Thermo Fisher Scientific (1 mentions)
Anti ifn γ, by Thermo Fisher Scientific (1 mentions)
Pe conjugated anti cxcr3, by BD (1 mentions)
Pe conjugated anti cd11b, by BD (1 mentions)
8 protocols using pe cy7 conjugated anti cd8
1
Flow Cytometry Analysis of T Cell Markers
2
Isolation and Characterization of CNS Mononuclear Cells
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For preparation of CNS mononuclear cells, brains and spinal cords from MOG35–55-immunized mice were excised and dissociated for 45 min at 37 °C by digestion with collagenase IV (2 mg/ml, Sigmal-Aldrich) and DNase I (100 μg/ml, Sigmal-Aldrich) in DMEM medium. Dispersed cells were passed through a 70 μm nylon mesh and collected by centrifugation. CNS mononuclear cells were isolated through a Percoll density gradient and collected from the interface fraction between 37% and 70% Percoll. After extensive washing, suspensions of cells were stained with FITC-labeled anti-CD4, PE-conjugated anti-CD11b, APC-conjugated anti-CD45, PE-Cy7-conjugated anti-MHC ΙΙ, PE-Cy7-conjugated anti-CD8, PE-conjugated anti-CXCR3, PE-conjugated anti-CCR5 (all from BD Pharmingen, San Diego, CA, USA). Isotype controls were used for determination of negative cells. The stained cells were analyzed on a FACSAria instrument (BD Bioscience, San Diego, CA, USA). For Th1, Th17 and Treg cell analysis, cells were stained with surface marker, permeabilized with the Intracellular Fixation and Permeabilization Buffer Set (eBioscience, San Diego, CA,USA), and then stained with anti IFN-γ, anti-IL17A, GM-CSF, and anti-Foxp3 antibodies. All these antibodies were purchased from eBioscience, unless marked otherwise.
3
Multi-parameter Flow Cytometry Analysis
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A CNS and spleen leukocyte suspension was obtained as described previously (Carrillo‐Salinas et al., 2017 (link)). Isolated cells were incubated with anti‐CD16/CD32 (Affymetrix Inc.) for FcR blockade and labeled with anti‐mouse antibodies: PE‐conjugated anti‐CD44 (2.4 μg/ml), PE‐conjugated anti‐CD274 (2.4 μg/ml), PerCP‐Cy5.5‐conjugated anti‐CD4 (1.2 μg/ml), PECy7‐conjugated anti‐Ly6c (1.2 μg/ml), APC‐Cy7‐conjugated anti‐CD11b (1.2 μg/ml), APC‐conjugated anti‐CD62L (2.4 μg/ml), APC‐conjugated anti‐CD25 (2.5 μg/ml), and APC‐conjugated anti‐CD1d (2.4 μg/ml; all from eBioscience); APC‐conjugated anti‐P2yR12 (2.5 μg/ml) and APC‐Cy7‐conjugated anti‐CD3 (1.2 μg/ml) (both from Biolegend); PE‐conjugated anti‐CD5 (2.2 μg/ml), PerCP‐Cy5.5‐conjugated anti‐B220 (2.4 μg/ml), PerCP‐Cy5.5‐conjugated anti‐CD45 (1.2 μg/ml), PECy7‐conjugated anti‐CD8 (1.2 μg/ml) and PECy7‐conjugated anti‐CD19 (2.4 μg/ml; all from BD Pharmingen). The cells were fixed for 30 min with fixation buffer (Affymetrix Inc.). For FoxP3 detection, the cells were suspended in Fixation/Permeabilization buffer for 30 mins prior to staining with an anti‐FoxP3 antibody (3 μg/ml; BD Pharmingen). At least 50,000 events were registered in each experiment on a FACSAria flow cytometer (BD Biosciences), excluding duplets from the analysis. The data were analyzed using FACSDiva analysis software (BD Biosciences).
4
Longitudinal Immunophenotyping of PBMC
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PBMC samples were obtained from patients before the administration of αGalCer-pulsed APCs, and another eight times during the 11-week study period (figure 1 ). mAbs to detect and fractionate mononuclear cells in peripheral blood were: FITC-conjugated anti-TCR Vα24 (Beckman Coulter, Brea, CA), anti-CD14 (BD Biosciences), anti-CD45RA (BD Biosciences), PE-conjugated anti-TCR Vβ11 (Beckman Coulter), anti-CD56 (BD Biosciences), anti-CCR7 (Biolegend), allophycocyanin-conjugated anti-CD3 (BD Biosciences), αGalCer-loaded CD1d tetramer (ProImmune), allophycocyanin-Cy7 conjugated anti-CD3 (BD Biosciences), PE-Cy7-conjugated anti-CD8 (BD Biosciences), PB-conjugated anti-CD4 (BD Biosciences), anti-CD16 (BD Biosciences), anti-PD-1 (Biolegend), and anti-HLA-DR(Biolegend). The absolute numbers of these cells were also calculated using automated full blood counts.
5
Immune Profiling of Residual Lymphoma in Mice
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At day 45 p.t.i. mice with residual primary lymphoma from CHOP×2 group (i.e. animals with partial response to chemotherapy) as well as PBS control, were sacrificed and tumors were removed and prepared as a single-cell suspension. Cells were immunostained at 4 °C in the dark for 30 min with the following panel of antibodies: FITC-conjugated anti-CD49b, PECy7-conjugated anti-CD8, APC-conjugated anti-CD3, APCCy7-conjugated anti-CD4, PerCPCy5.5-conjugated anti-CD19, FITC-conjugated anti-CD4, PE-conjugated anti-FoxP3, PECy7-conjugated anti-CD3, APC-conjugated anti-CD25, FITC-conjugated anti-Ly6C, PE-conjugated anti-Ly6G, (all reagents from BD Pharmingen, San Diego, CA). Optimal antibody concentration was previously defined by titration. For intracellular FoxP3 staining, cells were first stained with anti-CD4 and anti-CD25 antibodies, then fixed and permeabilized with a mouse FoxP3 buffer set (BD Pharmingen) according to the manufacturer’s protocols. Cells were washed twice with permeabilization buffer and then incubated with anti-FoxP3 at 4 °C for 30 min in the dark. Flow cytometry data were collected on a FACS Canto II flow cytometer equipped with two lasers (Becton–Dickinson, Oxford, UK). For data acquisition and analysis, FACSDiva (Becton–Dickinson) and Infinicyt (Cytognos, Spain) software were used.
6
Immunophenotyping by Flow Cytometry
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We directly incubated cells with fluorescent-labeled antibodies and performed cell fluorescence analysis using flow cytometry (BD FACS Canto) to ascertain cell phenotypes for assessment. CD8 and PD-L1 can be stained straight on the cell surface and discovered, while granular enzyme B and IFN-γ are employed for intracellular staining, so cells are foremost immobilized with 4% paraformaldehyde for 20-30 min, then stained with fluorescent-pigment-labeled antibodies, and incubated in dark ice for 15 min. Antibody choice FITC-conjugated anti-Granzyme B antibodies and anti-IFN-γ were stained with PE-conjugated anti-PD-L1, PE-Cy7-conjugated anti-CD8, and APC-conjugated cells (BD, USA).
7
Tumor Infiltrating Immune Cells Analysis
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At day 45 p.t.i., five mice from PBS and LVR01x3 groups and five mice with residual lymphoma (partial response) from CHOPx2 and CHOPx2 + LVR01x3 were sacrificed and tumors were removed and prepared to obtain a single-cell suspension. 1 × 105 cells per tumor were immunostained at 4°C in the dark for 30 min with the following antibodies panel: FITC-conjugated anti-CD49b, PECy7-conjugated anti-CD8, APC-conjugated anti-CD3, APCCy7-conjugated anti-CD4, PerCPCy5.5-conjugated anti-CD19, FITC-conjugated anti-CD4, PE-conjugated anti-FoxP3, PECy7-conjugated anti-CD3, APC-conjugated anti-CD25, FITC-conjugated anti-Ly6C, and PE-conjugated anti-Ly6G (all reagents from BD Pharmingen, San Diego, CA, USA). The optimal antibody concentration was defined by titration. For Treg cells analysis, cells were first stained with anti-CD4 and anti-CD25 antibodies, then fixed and permeabilized with a mouse FoxP3 buffer set (BD Pharmingen) and then washed twice with permeabilization buffer and incubated with anti-FoxP3 at 4°C for 30 min in the dark. Flow cytometry data were collected on a FACS Canto II Cytometer (Becton–Dickinson, Oxford, UK). For data acquisition and analysis, FACSDiva (Becton–Dickinson) and Infinicyt (Cytognos, Spain) software were used, respectively.
8
Flow Cytometry Antibodies for Mouse Immune Cell Analysis
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The following anti-mouse antibodies were obtained from BD PharMingen: PerCP-conjugated anti-CD3e (553067, 1 : 100), PE-conjugated anti-CD8 (557654, 1 : 100), PE-Cy7-conjugated anti-CD8 (552877, 1 : 100), APC-Cy7-conjugated anti-CD8 (557654, 1 : 100), PE-conjugated anti-CD90.1 (561404, 1 : 100), APC-conjugated anti-CD90.1 (557266, 1 : 100), PE-Cy7-conjugated anti-CD62L (560516, 1 : 100), APC-Cy7-conjugated CD62L (560514, 1 : 100), and APC-conjugated anti-IFN-γ (554413, 1 : 100). Biolegend: PE-conjugated anti-CD44 (103008, 1 : 100), PE-Cy7-conjugated anti-CD44 (103030, 1 : 100), PE-Cy7-conjugated anti-CD69 (104512, 1 : 100), and APC-conjugated anti-CD103 (121414, 1 : 100). Flow cytometry data were acquired with a FACS Canto II flow cytometer (BD Biosciences) and data were analyzed with Flowjo software (Tree Star).
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