The capacity of immature or mDC to stimulate an allogeneic T cells response was determined by mixed lymphocyte reaction assay. Briefly, immature or mDC derived in the presence or absence of hCPC, were co-cultured with allogeneic CFSE-labeled PBLs in 6-well plates (Falcon, Cat. 353046) for 5 days at 300,000 PBLs per 60,000 DC (ratio5:1). Proliferation of CD4+, CD8+ T cell subsets was monitored by flow cytometry using anti-CD3-APC, anti-CD4-Pacific blue, anti-CD8-APC-H7 (BD Biosciences), anti-CD25-PE (Miltenyi Biotec), and 7-AAD.
Anti cd25 pe
Manufactured by Miltenyi Biotec
Sourced in Germany
Anti-CD25-PE is a fluorescent-labeled antibody product designed for the detection and analysis of CD25-positive cells using flow cytometry. It binds specifically to the CD25 antigen expressed on the surface of activated T cells and regulatory T cells.
Automatically generated - may contain errors
Lab products found in correlation
Anti gag p19, by ZeptoMetrix (2 mentions)
6 well plate, by BD (1 mentions)
Anti cd3 apc, by BD (1 mentions)
7 aad, by Miltenyi Biotec (1 mentions)
Anti cd8 apc h7, by BD (1 mentions)
Anti cd4 pacific blue, by BD (1 mentions)
Rbc lysis buffer, by Thermo Fisher Scientific (1 mentions)
Anti cd3 viogreen, by Miltenyi Biotec (1 mentions)
Anti cd69 apcvio770, by Miltenyi Biotec (1 mentions)
Flowlogic software, by Inivai Technologies (1 mentions)
Brdu labeling and detection kit 3, by Roche (1 mentions)
Magnetic activated cell sorter immunomagnetic separation system, by Miltenyi Biotec (1 mentions)
Anti pe labeled microbeads, by Miltenyi Biotec (1 mentions)
Elisa microplate reader, by Agilent Technologies (1 mentions)
Opteia human ifn γ, by BD (1 mentions)
Opteia human tnf elisa kit, by BD (1 mentions)
Anti cd8 fitc, by Miltenyi Biotec (1 mentions)
Anti cd8 apc vio770, by Miltenyi Biotec (1 mentions)
Tim 3 bv421, by BioLegend (1 mentions)
Anti c myc fitc, by Miltenyi Biotec (1 mentions)
12 protocols using anti cd25 pe
1
Evaluating Allogeneic T Cell Response to mDC
2
Multiparameter Flow Cytometry Analysis
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Following 48 h, whole blood incubation, RBC lysis buffer (Thermo Fisher Scientific) was used to lyse the red blood cells. After washing with PBS, cells were stained with anti-CD3-Viogreen, anti-CD69-APCVio770, anti-CD95-PEVio770, anti-CD25-PE, anti-CD71-FITC, and anti-CD154-VioBlue (Miltenyi Biotec). The samples were measured after a final washing step, using a MaqsQuant10 analyzer. Before measurement, propidium iodide (PI) (Miltenyi Biotec) was added to assess viability. Analysis of the cell populations was performed with Flowlogic software (Inivai Technologies, Mentone VIC, Australia). For each time point the unstimulated samples were used to set the correct gating. The gating strategy is shown in Figure A1 in Appendix A .
3
Allogeneic T Cell Proliferation Assay
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Splenocytes (normalized to 1 × 105 T cells per well) from HSCT recipients were cultured in the presence or absence of irradiated (2,000 cGy) splenocytes (2 × 105 cells per well) from different mouse strains in a 96‐well plate for 4 days. Cell proliferation was measured by BrdU Labeling and Detection Kit III (Roche Applied Science, Mannheim, Germany,
https://lifescience.roche.com ) according to the manufacturer's instructions. Absorbance (measured as optical density [OD]), proportional to BrdU uptake, was measured at 405 nm using an ELISA microplate reader (BioTek, Winooski, VT,
http://www.biotek.com ). The data are expressed as stimulation index (OD in mixed leukocyte reactions [MLRs]/OD in spontaneous proliferation).
For one‐way MLRs, splenocytes from HSCT recipients were depleted for CD25+ T cells before MLR. Briefly, splenocytes were incubated with anti‐CD25‐PE, and then anti‐PE‐labeled microbeads (Miltenyi Biotec). Depletion of CD25+ cells was achieved by using a magnetic‐activated cell sorter immunomagnetic separation system (Miltenyi Biotec) as described29 . The purity of the depletion using this procedure, assessed by flow cytometry, was >97%.
For one‐way MLRs, splenocytes from HSCT recipients were depleted for CD25+ T cells before MLR. Briefly, splenocytes were incubated with anti‐CD25‐PE, and then anti‐PE‐labeled microbeads (Miltenyi Biotec). Depletion of CD25+ cells was achieved by using a magnetic‐activated cell sorter immunomagnetic separation system (Miltenyi Biotec) as described
4
T-cell Activation and Cytokine Profiling
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After 22 h-incubation of 5×103 PC3-PSCA tumor cells with 5×104 freshly isolated PBMCs or isolated CD8+ T cells in the presence or absence of 30 pmol/ml of recombinant Abs, cells were spun down and supernatants were collected. T cells of one triplet were pooled, stained with anti-CD25/PE, anti-CD69/PE-Cy5, and anti-CD8/FITC (Miltenyi Biotec GmbH) and measured using a flow cytometer. Cytokine concentrations in collected supernatants were determined using OptEIA Human IFN-γ and OptEIA Human TNF ELISA Kits (BD Biosciences).
5
T Cell Activation Markers Analysis
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Activation markers were analyzed by flow cytometry 14 h after starting the co-culture of T cells with target cells at a 1:1 E:T ratio. Anti-CD25-PE, anti-CD69-PE-Vio770, anti-CD137-APC, anti-CD8-APC-Vio770, anti-CD4-VioGreen, and anti-c-myc-FITC (all Miltenyi Biotec) and TIM-3-BV421 (Biolegend) were used. Surface marker stains were analyzed using a MACSQuant Analyzer 10 (Miltenyi Biotec).
6
Multiparametric Analysis of T-cell Subsets
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PBMCs were thawed, washed with PBS, and stained with the Zombie Yellow Fixable Viability Kit for 25 min at room temperature. Subsequently, the cells were washed with PBS and incubated for 25 min at 4°C with antibodies for membrane marker staining prediluted in flow cytometry buffer: anti-CD3 PE/Dazzle 594, anti-CD4 FITC, anti-CD45RA PE/Cy7, anti-CD27 Brilliant Violet 421, anti-inducible T-cell costimulator (ICOS) Brilliant Violet 421, anti-CD127 Brilliant Violet 510, anti-C-C chemokine receptor type 7 (CCR7) PE/Cy7, anti-HLA-DR PerCP, anti-CD62L PerCP/Cyanine 5.5 (all from Biolegend), and anti-CD25 PE (Miltenyi Biotec). Afterwards, the cells were washed with flow cytometry buffer and fixation/permeabilization reagent (Foxp3/transcription factor buffer set, eBioscience) was added to each sample followed by an incubation step of 25 min at 4°C. Then, the cells were washed with permeabilization buffer (Foxp3/transcription factor buffer set) and incubated with anti-Foxp3 APC (eBioscience), prediluted in permeabilization buffer, for 25 min at 4°C. Finally, cells were washed with permeabilization buffer and resuspended in flow cytometry buffer. Acquisition and compensation was performed as described for ICS.
7
Suppression of Effector T Cells by iTregs
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iTregs were generated from TxA23-Thy1.1/1.1 mice. 5×106 cells were injected I.P. into 3 TxA23-Thy1.1/1.1 mice and 3 TxA23-Thy1.1/1.1 mice were injected with PBS. Two days after iTreg injection, 1×106 CFSE labeled effector T cells were injected I.V. into all mice. Effector T cells were isolated from the gastric lymph node and spleen of TxA23-Thy1.1/1.2 mice using Miltenyi AutoMACS beads. Gastric lymph node and spleen were processed into a single cell suspension. Total T cells were isolated using a PAN-TII isolation kit (Miltenyi). Tregs in the spleen and gastric lymph node were removed by labeling cells with an anti-CD25-PE and then using anti-PE beads (Miltenyi). The remaining CD4 cells were then labeled with Carboxyfluorescein succinimidyl ester (CFSE) proliferation dye prior to injection. One week after injection of effector T cells, the gastric lymph node was isolated from both iTreg treated and PBS treated groups. Proliferation of transferred Thy1.2+ effector T cells was determined by dilution of CFSE dye using Flow Cytometry.
8
Quantifying Retroviral Infection Rates
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106 MT-2 cells were co-cultured with 106 Jurkat T-cells for 1h at 37°C. After washing with PBS, cells were fixed with 2% PFA (15min, 25°C) and permeabilized using saponin: cells were incubated with mouse monoclonal antibodies anti-gag p19 (ZeptoMetrix Corporation) in PBS/0.3% saponin (30min, 4°C) and washed with PBS/0.1% saponin. Cells were incubated with anti-mouse AlexaFluor647-conjugated secondary antibodies (LifeTechnologies GmbH) in PBS/0.3% saponin (30min, 4°C) and washed twice with PBS/0.1% saponin. After washing with PBS/5% FCS, cells were incubated with anti-CD25-PE (Miltenyi Biotech GmbH, Bergisch-Gladbach, Germany) in PBS/5% FCS to stain MT-2 cells (10min, 4°C). Cells were analyzed using the BD LSR II flow cytometer (BD Biosciences). Cells were discriminated by their different size (FSC/SSC) and by CD25-staining. The percentage of gag-positive cells within CD25-negative cells (Jurkat T-cells) was examined to measure gag transfer and infection rates from MT-2 to Jurkat T-cells.
9
Multiparametric Flow Cytometry of HDAC9
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HDAC9 expression was evaluated by a multiparametric flow cytometry analysis in 5 HT, 5 GD, and 6 healthy controls in different cell subsets by using anti-CD3-PE (BD Biosciences Cat# 555333, RRID:AB_395740); anti-CD4-PerCP (BD Biosciences Cat# 345770, RRID:AB_2868798); anti-CD25-PE (Miltenyi Biotec Cat# 130-113-286, RRID:AB_2733792); anti-CD14-FITC (BD Biosciences Cat# 555397, RRID:AB_395798); anti-CD19-PE (BD Biosciences Cat# 555413, RRID:AB_395813); IL-17-APC (Thermo Fisher Scientific Cat# 17-7179-42, RRID:AB_1582221); HLA-DR-PerCP (BD Biosciences Cat# 347364, RRID:AB_400292); CD11c-Pacific Blue (BD Biosciences Cat# 560369, RRID:AB_1645557); BDCA1-PE (BD Biosciences Cat# 564900, RRID:AB_2739006) monoclonal antibodies. Intracellular stainings were performed following permeabilization with 0.3% saponin. In addition, cells were fixed and permeabilized with the FOXP3 Fix/Perm kit (eBioscience) and stained with anti-Foxp3-FITC (Thermo Fisher Scientific Cat# 11-4777-42, RRID:AB_11149498) and anti-HDAC9 (Abcam Cat# ab109446, RRID:AB_10861804) followed by an Alexa Fluor 647 (Abcam Cat# ab150075, RRID:AB_2752244). Data were acquired on a FACS Canto II flow cytometer (BD Biosciences), and data were analyzed by using the FlowJo software v7.6 (Tree Star).
10
Regulatory T Cell Induction Protocol
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For in vitro regulatory T cell induction, CD4+ T lymphocytes were seeded in wells pretreated with 1 μg/mL anti-CD3 (Thermo Fisher Scientific, San Diego, CA, USA) and then stimulated with 25 µL of Dynabeads Human T-Activator CD3/CD28 (Life Technologies AS, Norway), 5 ng/mL TGF-β, and 0.1 μM/mL all-trans-retinoic acid (Merck Life Science, Madrid, Spain) for 2 days with T lymphocytes alone or in the presence of MSCs at a ratio of 1:10 (MSCs: CD4+). After 2 days, cells were stained with the following antibodies: Anti-CD4-VioBlue and anti-CD25-PE or with the corresponding isotype control antibodies following the manufacturer’s instructions (Miltenyi Biotec, Bergisch Gladbach, Germany). Then, cells were fixed and permeabilized using FoxP3 Staining Buffer Set (Miltenyi Biotec, Bergisch Gladbach, Germany). Finally, T lymphocytes were stained with anti-FoxP3-APC or with the corresponding isotype control antibodies. Data were acquired using the MACSQuant® analyzer (Miltenyi Biotec, Bergisch Gladbach, Germany). Data were analyzed using FlowJoTM v10.6.2 Software (BD Life Sciences, Ashland, OR, USA).
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