Flow cytometry was performed as previously described [38 (link)]. Briefly, cells were incubated with LIVE/DEAD Fixable cell stain (Invitrogen), then stained for 20 min with: MHCII-PacificBlue, F4/80-APC (Biolegend), CD4-AF700, CD8-PacificBlue, CD25-APC, CD86-APC, CD49b-Pe-Cy7 (eBioscience), CD11b-PE-Cy7, CD45R/B220-PerCP-Cy5.5 (BD-Pharmingen). For intracellular staining, the samples were stimulated for 3 h (100 ng/ml phorbol 12-myristate 13-acetate, 1 μg/ml ionomycin; monensin added after 1 h). Cells were fixed and permeabilized with Cytofix/Cytoperm (BD), then stained with IL-10-FITC, IFN-γ-PerCP-Cy5.5, IL-17-PE, FoxP3-AF488 (BD-Pharmingen) antibodies before analysis on LSRII flow cytometer (BD), collecting 100,000 live events. Data was analyzed using FlowJo (v7.6.5, Tree Star, Ashland, OR, USA).
Cd25 apc
Manufactured by Thermo Fisher Scientific
Sourced in United States
CD25-APC is a fluorescently labeled antibody that binds to the CD25 protein, also known as the IL-2 receptor alpha chain. It is used in flow cytometry applications to identify and quantify cells expressing the CD25 marker.
Automatically generated - may contain errors
Lab products found in correlation
Cd4 fitc, by Thermo Fisher Scientific (26 mentions)
Foxp3 pe, by Thermo Fisher Scientific (18 mentions)
Cd4 percp, by BD (5 mentions)
Facscalibur flow cytometer, by BD (5 mentions)
Lsrii flow cytometer, by BD (4 mentions)
Facscalibur, by BD (4 mentions)
Cytofix cytoperm, by BD (3 mentions)
Foxp3 pe, by BioLegend (3 mentions)
Il 17a apc, by Thermo Fisher Scientific (3 mentions)
Cd3 fitc, by Thermo Fisher Scientific (3 mentions)
Cd3 efluor450, by Thermo Fisher Scientific (3 mentions)
Il 17a pe, by Thermo Fisher Scientific (3 mentions)
Cd25 pe, by Thermo Fisher Scientific (3 mentions)
Cd11b pe cy7, by Thermo Fisher Scientific (3 mentions)
Fcr blocking reagent, by Miltenyi Biotec (3 mentions)
Cd8 pacific blue, by Thermo Fisher Scientific (2 mentions)
Cd4 percp cy5, by BioLegend (2 mentions)
Live dead fixable blue dead cell stain kit, by Thermo Fisher Scientific (2 mentions)
Cd206 apc, by BioLegend (2 mentions)
Cd8 alexa fluor 700, by BD (2 mentions)
58 protocols using cd25 apc
1
Comprehensive Immune Cell Profiling by Flow Cytometry
2
Multi-Color Flow Cytometry Immunophenotyping
3
Comprehensive Lung Cell Immunophenotyping
4
Phenotypic analysis of lymphocyte subsets
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The cells were incubated with anti‐CD16/CD32 (KT1632, Invitrogen, Thermo Fisher Scientific) for 15 minutes at 4 ℃. Single‐cell suspensions of splenocytes and peripheral blood mononuclear cells were stained with CD4 fluorescein isothiocyanate (100510, BioLegend, San Diego, CA, USA) and CD25 PE (102007, BioLegend). Mononuclear cells isolated from the brains were stained with CD3e PerCP‐Cyanine5.5 (45‐0031‐82, eBioscience, San Diego, CA, USA), CD4 fluorescein isothiocyanate (11‐0041‐82, eBioscience, ), CD8a PE‐Cyanine7 (25‐0081‐82, eBioscience), and CD25 APC (17‐0257‐42, eBioscience) for 30 minutes at 4℃. Intracellular staining with Foxp3 APC (17‐5773‐82, eBioscience) for splenocytes and peripheral blood mononuclear cells and Foxp3 PE (12‐4777‐42, eBioscience) for mononuclear cells isolated from brains was followed by incubation for 30 minutes at 4℃ after cell fixation and permeabilization using the Foxp3/Transcription Factor Staining Buffer Set (562574, BD Pharmingen Inc., CA, USA) according to the manufacturer’s instructions. Finally, data were acquired with an LSRII cytometer (BD Biosciences, San Jose, CA, USA) and analysis was performed using FlowJo (TreeStar, Ashland, OR, USA).
5
Comprehensive Immunological Signaling Analysis
6
Regulatory T Cell Phenotyping
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Fluorochrome-labeled monoclonal antibodies mainly CD4-FITC, CD127-PE, and CD25-APC (eBioscience, San Diego, USA), which identified Tregs, were added to flow measuring tubes. Fifty microliters of whole blood samples treated with EDTA-K3 were added to the tube, mixed well, and incubated for 20 min at room temperature in the dark. Then, the samples were added with 2 ml RBC lysing buffer and mixed well to lyse RBC, incubated for 10 min at room temperature in the dark, and centrifuged, after which the supernatant was discarded. Later, they were washed with 2 ml phosphate buffered saline containing 0.5% bovine serum albumin and centrifuged at 1,000 rpm for 5 min, after which the supernatant was discarded. The cells were resuspended in 300 μl fixative solution and placed at 4°C in the dark. Data were acquired by flow cytometry; fixed cells were analyzed using CellQuest/Diva software within 24 h; and the percentage of CD4+CD25+CD127lo/− cells was obtained in CD4+ lymphocytes.
7
Comprehensive Immunophenotyping of T and B Cells
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Phenotypic analyses of T cells and B cells were performed with anti-human monoclonal antibodies (mAbs): anti-human CD3-PerCP, CD4-FITC, CD19-PerCP and CD21-APC were from BD Biosciences (San Jose, CA, USA). CXCR5-APC, ICOS-PE, PD-1-PE, IFN-γ-PE, IL-4-PE, IL-17-PE, IL-21-PE, IL-22-PE, CD27-FITC, CD86-PE, CD95-PE, CD25-APC, and Foxp3-PE antibodies were obtained from eBiosciences (San Diego, CA, USA). Cells were analyzed by flow cytometry (BD FACSCalibur, San Jose, CA) and data was analyzed by FlowJo software (Tree Star, San Carlos, CA).
8
Murine Intracellular Cytokine Staining
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Briefly, mouse spleen and draining lymph node (dLN) and kidney grinded to cell suspension resuspended in a red blood cell lysing buffer at 37°C for 2 min, followed by passing through a 70 mm cell strainer and centrifuged.
For intracellular IL-17A staining, cells were treated with 5 ng/ml PMA (Invitrogen) and 1 ng/ml ionomycin (Enzo Life Sciences, Inc.) for 5 h and then added at 10 ng/ml brefeldin A (Enzo Life Sciences, Inc.) 30 min before staining with CD4- FITC (eBiosciences, San Diego, United States). Next cells were stained with IL-17A-PE (eBiosciences) followed by permeabilization of the cells with Cytofix/Cytoperm (BD Biosciences, San Diego, United States).
For intracellular Foxp3-PE staining (Pan et al., 2014 (link)), cells were permeabilized with Cytofix/Cytoperm, and stained with Foxp3-PE (eBiosciences), followed by stain with CD4-FITC and CD25-APC (eBiosciences). Cells were detected by FACS Calibur Flow Cytometer (Becton Dickinson, San Diego, United States) and analyzed with the Flow Jo software.
For intracellular IL-17A staining, cells were treated with 5 ng/ml PMA (Invitrogen) and 1 ng/ml ionomycin (Enzo Life Sciences, Inc.) for 5 h and then added at 10 ng/ml brefeldin A (Enzo Life Sciences, Inc.) 30 min before staining with CD4- FITC (eBiosciences, San Diego, United States). Next cells were stained with IL-17A-PE (eBiosciences) followed by permeabilization of the cells with Cytofix/Cytoperm (BD Biosciences, San Diego, United States).
For intracellular Foxp3-PE staining (Pan et al., 2014 (link)), cells were permeabilized with Cytofix/Cytoperm, and stained with Foxp3-PE (eBiosciences), followed by stain with CD4-FITC and CD25-APC (eBiosciences). Cells were detected by FACS Calibur Flow Cytometer (Becton Dickinson, San Diego, United States) and analyzed with the Flow Jo software.
9
Isolation and Characterization of Liver Leukocytes
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Liver leukocytes were isolated by mechanical dissociation as described previously18 (link). The following fluorescently-labeled antibodies targeting mouse antigens were used: CD45-BUV (BD Biosciences, Franklin Lakes, NJ, USA, 30-F11), CD3-FITC (BioLegend, San Diego, CA, USA, 17A2), NK1.1-APC-eFluor780 (eBioscience, PK136), CD25-APC (ebioscience, PC61.5), and CD69-PeCy7 (BD Biosciences, H1.2F3). Cells were acquired on a SORP LSRII (BD Pharmingen Inc., San Diego, CA) and flow cytometry data were analyzed using FlowJo software (FlowJo, LLC) following the gating strategy given in Supplementary Fig. 1 . The results were analyzed and displayed with GraphPad Prism software, representing the mean and standard deviation and analyzed using two-sided Student’s t-test. (GraphPad Software Inc., La Jolla, CA, USA).
10
Tumor Dissociation and Immune Cell Analysis
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Tumors were minced into thin pieces and were dissociated in DNase I (1 U/ml; Roche, Indianapolis, IN, USA), collagenase D (1 mg/ml; Roche), and 0.125% trypsin-EDTA (Gibco) in pre-warmed DMEM (Welgene) for an hour at 37 °C with gentle agitation. The tissues were mechanically dissociated on a 100 μm nylon mesh strainer. Next, the single cells were passed through a 40 μm nylon mesh strainer. Spleens were also mechanically dissociated on 40 μm nylon mesh strainer. RBCs were lysed for 5 minutes in 1X Pharmlyse buffer.
Cells were stained with fluorescently tagged antibodies. All data were detected by a FACSCalibur cytometric system and analyzed by FlowJo software. The following fluorophore-labeled antibodies were purchased from e-bioscience (San Diego, CA, USA): CD45-FITC, CD3-FITC, CD4-PE, CD4-FITC, CD8-APC, CD11b-APC, CD11c-PE, CD25-PE, CD25-APC, B220-FITC, CD19-PE, CD86-APC. Foxp3-Alexa Fluor647 was purchased from BD bioscience, and F4/80-PE and CD206-APC were obtained from Biolegend (San Diego, CA, USA).
Cells were stained with fluorescently tagged antibodies. All data were detected by a FACSCalibur cytometric system and analyzed by FlowJo software. The following fluorophore-labeled antibodies were purchased from e-bioscience (San Diego, CA, USA): CD45-FITC, CD3-FITC, CD4-PE, CD4-FITC, CD8-APC, CD11b-APC, CD11c-PE, CD25-PE, CD25-APC, B220-FITC, CD19-PE, CD86-APC. Foxp3-Alexa Fluor647 was purchased from BD bioscience, and F4/80-PE and CD206-APC were obtained from Biolegend (San Diego, CA, USA).
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