Various subpopulations within the cell products (PBMCs prior to culture and cultured DC-CIKs) were identified by flow cytometry, as previously described (11 (link)), using the following fluorochrome-conjugated antibodies: CD3 PerCP-Cy5.5, CD4 FITC, CD8 FITC, CD25 PE, CD28 PE, CD56 PE (all Beckman Coulter, Inc.), PD-1 PE, lymphocyte-activation gene 3 (LAG-3) PE, tumor necrosis factor receptor superfamily member 9 (4-1BB; CD137) PE and T cell immunoglobulin and mucin protein 3 (TIM-3) PeCy-7 (all BioLegend, Inc.). Three-color flow cytometry analysis was performed on a Cytomics FC500 flow cytometer with CXP analysis software (Beckman Coulter, Inc.).
Cd25 pe
Manufactured by Beckman Coulter
Sourced in United States
The CD25-PE is a flow cytometry reagent that detects the expression of CD25, a cell surface marker. It is used to identify and quantify activated T cells in biological samples. The CD25-PE reagent consists of a fluorescent dye (phycoerythrin) conjugated to an anti-CD25 antibody.
Automatically generated - may contain errors
Lab products found in correlation
Cxp analysis software, by Beckman Coulter (4 mentions)
Cd4 fitc, by Beckman Coulter (3 mentions)
Cd8 fitc, by Beckman Coulter (3 mentions)
Cd3 fitc cd16 cd56 pe, by Beckman Coulter (3 mentions)
Cd19 pc5, by Beckman Coulter (3 mentions)
Fc500, by Beckman Coulter (2 mentions)
Cd3 pc5 cd4 fitc cd8 pe, by Beckman Coulter (2 mentions)
Cd3 percp cy5, by Beckman Coulter (1 mentions)
Cd56 pe, by Beckman Coulter (1 mentions)
Lag 3 pe, by BioLegend (1 mentions)
Cd137 pe, by BioLegend (1 mentions)
Tim 3 pe cy7, by BioLegend (1 mentions)
Cd28 pe, by Beckman Coulter (1 mentions)
Cytomics fc500 flow cytometer, by Beckman Coulter (1 mentions)
Specific monoclonal antibodies, by Beckman Coulter (1 mentions)
Cd45 fitc, by Beckman Coulter (1 mentions)
Cd38 pe, by Beckman Coulter (1 mentions)
Cd27 pe, by Beckman Coulter (1 mentions)
Cd8 pe, by Beckman Coulter (1 mentions)
Cytoflex flow cytometer, by Beckman Coulter (1 mentions)
10 protocols using cd25 pe
1
Phenotypic Characterization of Cell Subsets
2
Immunophenotyping of Lymphocyte Subsets
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Blood samples were collected at W0, W8, W24 and W48 for immunophenotyping by flow cytometry in ethylenediaminetetraacetic acid (8.55 mg/tube). Peripheral blood samples (100 µL) were incubated with specific monoclonal antibodies (Beckman Coulter, Wycombe, UK) at room temperature (RT) for 15 min in the dark; afterward, the red cells were lysed, at RT in the dark, in a VersaLyse lysing solution (A09777, Beckman Coulter, Wycombe, UK) for 15 min and then analysed. LS were identified by the recognition of surface molecules belonging to the CD family. The samples successively underwent gating analysis (total lymphocytes and CD45+ vs. side scatter) (CD45-FITC, A07782, Beckman Coulter, Wycombe, UK), and the desired lymphocyte subpopulations were gated excluding doublets. The LS identification was based on monoclonal antibodies against Bmem CD19+/CD27+, Br1 CD19+/CD38+, Br2 CD19+/CD25+, Treg CD4+/CD25+, Th CD3+/CD4+ and Tc CD3+/CD8+ (CD19-PC7, IM3628; CD27-PE, IM2578; CD38-PE, AO7779; CD25-PE, A07774; CD4-FITC, A07750; CD3-PC5, A07749; CD8-PE, A07757; Beckman Coulter, UK). The samples were tested using a CytoFLEX flow cytometer and the CytExpert software (Beckman Coulter, Wycombe, UK).
3
Immunophenotyping of Whole Blood Cells
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Whole blood samples (200 µL) were incubated with conjugated antibodies as indicated in the dark for 10 minutes at room temperature before red blood cells were lysed. Samples pellets were re-suspended in 500 µL phosphate-buffered saline (PBS) after centrifugation for 5 minutes at 1,300 g at room temperature and analyzed by flow cytometry.
Conjugated antibodies used in this study were purchased from Beckman Coulter (Brea, CA, USA) and included CD3-PC5/CD4-FITC/CD8-PE (IM1650), CD3-FITC/(CD16+/CD56)-PE (A07735), CD(14+16)-FITC/CD85k(ILT3)-PE/CD33-PC5 (A23413), CD4-FITC (A007750), CD8-FITC (A07756), CD19-PC5 (A07771), and CD25-PE (A07774).
Beckman-Coulter FC500 (Beckman Coulter, USA) and CXP analysis software (Beckman Coulter) was used for flow cytometry. There are 10,000 gated events in every analysis. Lymphocytes subtypes were selected according to physical characteristics including volume and transmissivity. The level of T lymphocyte subtype was expressed as percentage of the total number of lymphocytes.
Conjugated antibodies used in this study were purchased from Beckman Coulter (Brea, CA, USA) and included CD3-PC5/CD4-FITC/CD8-PE (IM1650), CD3-FITC/(CD16+/CD56)-PE (A07735), CD(14+16)-FITC/CD85k(ILT3)-PE/CD33-PC5 (A23413), CD4-FITC (A007750), CD8-FITC (A07756), CD19-PC5 (A07771), and CD25-PE (A07774).
Beckman-Coulter FC500 (Beckman Coulter, USA) and CXP analysis software (Beckman Coulter) was used for flow cytometry. There are 10,000 gated events in every analysis. Lymphocytes subtypes were selected according to physical characteristics including volume and transmissivity. The level of T lymphocyte subtype was expressed as percentage of the total number of lymphocytes.
4
EGFRvIII/CD3 TandAb-induced T-cell Activation
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To assess whether EGFRvIII/CD3 TandAb-induced activation of T-cells is dependent on the presence of target cells, primary human PBMC (4 × 105/well) were cultured in the presence of increasing concentrations EGFRvIII/CD3 TandAbs in the absence of EGFRvIII-positive target cells. After 5 days incubation, proliferation was assessed in a BrdU incorporation assay as previously described (28 (link)).
T-cell activation marker induction by EGFRvIII/CD3 TandAb was analyzed by FACS: primary human T-cells were cultured for 24 h with or without 10 µg/mL of TandAb in the presence or absence of F98EGFRvIII target cells at an E:T ratio of 1:1 before T-cells were analyzed by flow cytometry for expression of the activation markers CD25, CD137, and CD69 after staining of the cell cultures with CD4-FITC/CD8-FITC (Beckman-Coulter) in combination with CD25-PE (Beckman-Coulter), CD137-APC (BD Biosciences), or CD69-APC-Cy7 (BD Biosciences). As a control, cells were cultured in the absence of antibodies and in the absence of target cells.
T-cell activation marker induction by EGFRvIII/CD3 TandAb was analyzed by FACS: primary human T-cells were cultured for 24 h with or without 10 µg/mL of TandAb in the presence or absence of F98EGFRvIII target cells at an E:T ratio of 1:1 before T-cells were analyzed by flow cytometry for expression of the activation markers CD25, CD137, and CD69 after staining of the cell cultures with CD4-FITC/CD8-FITC (Beckman-Coulter) in combination with CD25-PE (Beckman-Coulter), CD137-APC (BD Biosciences), or CD69-APC-Cy7 (BD Biosciences). As a control, cells were cultured in the absence of antibodies and in the absence of target cells.
5
Multiparametric flow cytometry analysis
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CIKs and MSCs were harvested, transferred into PBS, and incubated with Fc-solution (Beckman Coulter) for 5 min at 4°C to prevent unspecific antibody binding. During the incubation with Fc-blocking reagent, antibodies were placed in fluorescence-activated cell sorter (FACS) tubes (Greiner, BIO). The following antibodies, purchased from Beckman Coulter (final concentrations in brackets), were used: CD3-FITC (1∶100), CD4-PE (1∶250), CD8a-PE (1∶250), CD11b-PE (1∶160), CD19-PE (1∶250), CD25-PE (1∶250), CD45-PE (1∶250), CD49-PE (1∶250), NK1.1-PE (1∶60), CD69-PE (1∶250), HamIgG-FITC-isotype-control (1∶250), and RatIgG2a-PE-isotype-controll (1∶250). In addition, CD34-PE (Caltaq, 1∶100), CD44-AF488 (Biozol, 1∶250), CD73-PE (Biozol, 1∶100), CD90 (Thy1.2)-FITC (Miltenyi, 1∶100), and CD166 (Antikörper-online, 1∶100).
After incubation for 5 min, Fc-blocked cells were added to the antibody-containing FACS tubes, incubated for 20 min at 4°C, and washed with PBS. Then, 7-aminoactinomycin-D (7-AAD; 1∶100, Beckman Coulter) stain was added, and the cells were incubated for 5 min at 4°C. Either isotypes or autofluorescence served as controls. Cells were gated on vital cells, indicated by low 7-AAD signals.
After incubation for 5 min, Fc-blocked cells were added to the antibody-containing FACS tubes, incubated for 20 min at 4°C, and washed with PBS. Then, 7-aminoactinomycin-D (7-AAD; 1∶100, Beckman Coulter) stain was added, and the cells were incubated for 5 min at 4°C. Either isotypes or autofluorescence served as controls. Cells were gated on vital cells, indicated by low 7-AAD signals.
6
Multiparameter Immune Cell Profiling
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Using Beckman Coulter's custom design service and its dry coating technology, custom tubes containing IFNγ-FITC (Clone 45.15), CD25-PE (Clone B1.49.9), CD4-PE-Cyanine5.5 (Clone 13B8.2), IL-4-PE-Cyanine7 (Clone MP4-25D2), Foxp3-AF647 (Clone 259D), TNFα-AF700 (Clone IPM2 (188)), CD3-AA750 (Clone UCHT1), IL-17A-PacBlue (Clone B168) and CD8-KromeOrange (B9.11) were produced.
7
Peripheral blood T-cell phenotyping
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Peripheral blood (4 ml) was obtained from each patient. Whole blood (200 μl) was incubated with primary antibody in the dark for 10 min at room temperature, and then samples went through hemolysis for another 10 min. Samples were then centrifuged for 5 min at 1300 g at room temperature, and the supernatant was removed. Finally, samples were re-suspended in 500 μl PBS and subjected to flow-cytometric analysis. Primary antibodies included CD3-PC5/CD4-FITC/CD8-PE (IM1650), CD3-FITC/(CD16+/CD56)-PE (A07735), CD (14 + 16)-FITC/CD85k (ILT3)-PE/CD33-PC5 (A23413), CD4-FITC (A007750), CD8-FITC (A07756), CD19-PC5 (A07771), and CD25-PE (A07774) (all from Beckman Coulter). Level of T lymphocyte subtype was expressed as percentage of the total lymphocytes. Total lymphocytes were selected according to physical characteristics including volume size and transmissivity. Flow cytometry was performed using Beckman-Coulter FC500 (Beckman Coulter, Inc., CA., US) and CXP analysis software (Beckman Coulter, Inc., CA., US). Peripheral cytotoxic T lymphocytes (pCTLs) were identified as CD8+ and CD28+. Each analysis included 10000 gated events.
8
Profiling Immune Cell Populations in PBMCs
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We collected peripheral blood mononuclear cells (PBMCs) through Ficoll density gradient centrifugation (Ficoll-Paque Premium; GE Healthcare). After they were resuspended with staining buffer (1× PBS with 0.5% bovine serum albumin and 0.5 mM EDTA), 2.5 × 10 5 PBMCs were labeled with the following cell surface markers: CD4 PE-Cy7 (SFCI12T4D11), CD8 Pacific Blue (RPA-T8), CD56 APC-Alexa Fluor 700 (N901(NKH-1)), CD3 Krome orange (UCHT1), CD14 APC-Alexa Fluor 750 (RMO52), CD19 APC-Alexa Fluor 750 (J3-119) (Beckman Coulter, Brea, CA); CD25 PE (BC96), CD11c APC (3.9), PD-1 Alexa flour 488 (EH12.2H7), programmed death-ligand 1 PE (PD-L1 PE) (29E.2A3), T cell receptor (TCR) α/β (TCR α/β FITC) (IP26), and TCR γ/δ APC (B1) (BioLegend, San Diego, CA). Some samples were further permeabilized and stained with antihuman Foxp3 APC (236A/E7) (eBioscience, San Diego, CA) and anti-CTLA-4 PE (L3D10) (BioLegend) antibodies. Data were acquired using a Navios flow cytometer (Beckman Coulter) and were analyzed using Kaluza software version 1.3 (Beckman Coulter).
9
Multiparameter Flow Cytometry Analysis
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Peripheral blood samples (200 μL) were incubated with anti-human antibody cocktails in the dark for 10 min at room temperature, and samples were then subjected to hemolysis for an additional 10 min. The cells were centrifuged for 5 min at 1300× g at room temperature and resuspended in 500 μL phosphate-buffered saline for flow cytometry. Antibody cocktails included CD3-PC5/CD4-FITC/CD8-PE (IM1650), CD3-FITC/CD16/CD56-PE (A07735), CD14/16-FITC/CD85k (ILT3)-PE/CD33-PC5 (A23413), CD4-FITC (A007750), CD8-FITC (A07756), CD19-PC5 (A07771), and CD25-PE (A07774) (all from Beckman Coulter, Brea, CA, USA). Flow cytometry was performed using an FC500 and CXP analysis software (both Beckman Coulter). Each analysis included 10,000 gated events.
10
Immune Cell Stimulation and Analysis
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For pharmacological stimulation, we used phorbol 12-myristate 13-acetate (PMA; Sigma) and ionomycin (Calbiochem). For antibody (Ab)-based stimulation, we used mouse antihuman or hamster antimouse monoclonal anti-CD3 and -CD28 Ab (555336, 555725, 553058, 553295; all from BD Pharmingen). For flow cytometry analyzes, we used LIVE/DEAD fixable red cell death staining kit (L23102, Invitrogen) and antimouse CD45-APC (L23102, eBioscience) or Percp-Cy5.5, CD8-BV610 and CD25-PCy7 (103132, 100744, and 102016, respectively; all from Biolegend), TCRβ-FITC (732247, Beckman Coulter) and PD-1-APC780 (47998582, eBioscience), antihuman PD-1-APC (17-9969-42, eBioscience) and CD25-PE (A07774, Beckman Coulter) and paraformaldehyde (TedPella). For western blot analysis, we used anti-DGKα (11 547–1-AP, ProteinTech), DGKζ (105195, Abcam), α tubulin (T5168, Sigma), GAPDH (G-9, Santa Cruz), horseradish peroxidase-conjugated antimouse and rabbit IgG (P0447, P0260, Dako), antirabbit IgG Dylight 800 (SA5-35571, Invitrogen) and mouse IgG AlexaFluor 680 (175775, AbcamLife). IKKβ inhibitor PS-1145 was from Sigma-Aldrich, MEK inhibitor PD98059 was from Calbiochem, and calcineurin (CaN) inhibitor FK506 was from Merck Millipore. Anti-PD-1 (nivolumab) humanized Ab was from BioVision. For TIL isolation, we used collagenase type I (Worthington), dispase II and DNase I (both from Roche). Trypsin was from Biowest.
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