Cell surface and intracellular molecular expressions were evaluated by flow cytometry using CytoFLEX (Beckman Coulter, USA). Fluorescein-conjugated anti-mouse antibodies, including CD3-BV785/BV510/AF700, CD4-BV510/BV605, CD25-FITC, FOXP3-PE, IL-10-APC, TGF-β1-BV421 (Biolegend, UK), and anti-human antibodies, including CD3-BV510, CD4-BV650, CD25-APC, FOXP3-PE, IL-10-BV421, TGF-β1-PE/CY7 (Biolegend, UK), were used according to the manufacturer's procedures. For cell-surface staining, single-cell suspensions were stained on ice for 30min in PBS with 1% FBS. For intracellular staining, cells were fixed and permeabilized using the Fix/Perm kit (Biolegend, UK). To detect intracellular cytokines, cells were stimulated for 4 h with Cell Activation Cocktail (with Brefeldin A) (Biolegend, UK). Thereafter, cells were harvested, stained for surface expression, and then fixed and permeabilized for intracellular staining. Flow cytometry data were analyzed using FlowJo software (BD, UK).
Foxp3 pe
Manufactured by BioLegend
Sourced in United States, United Kingdom
Foxp3-PE is a fluorescent-conjugated antibody that binds to the transcription factor Foxp3, a key regulator of regulatory T cells. It can be used for the identification and analysis of Foxp3-expressing cells by flow cytometry.
Automatically generated - may contain errors
Lab products found in correlation
Cd25 apc, by BioLegend (13 mentions)
Cd4 fitc, by BioLegend (11 mentions)
Cd3 fitc, by BioLegend (6 mentions)
Cd4 pe cy7, by BioLegend (6 mentions)
Cd25 fitc, by BioLegend (4 mentions)
Cd3 pe cy7, by BioLegend (4 mentions)
Cd25 apc, by Thermo Fisher Scientific (3 mentions)
Cd4 fitc, by Thermo Fisher Scientific (3 mentions)
Cd8a apc, by BioLegend (3 mentions)
Cd8 fitc, by BioLegend (3 mentions)
Cytofix cytoperm kit, by BD (3 mentions)
Cd8 apc, by BioLegend (3 mentions)
Rbc lysis buffer, by Thermo Fisher Scientific (3 mentions)
Anti mouse cd16 32, by Thermo Fisher Scientific (3 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (3 mentions)
Cd86 pe, by BioLegend (3 mentions)
F4 80 fitc, by BioLegend (3 mentions)
Fix perm kit, by BioLegend (2 mentions)
Lsr 2 flow cytometer, by BD (2 mentions)
F4 80 pe, by BioLegend (2 mentions)
44 protocols using foxp3 pe
1
Evaluating Immune Cell Phenotypes
2
Comprehensive Immune Cell Profiling
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The heparinised whole blood was withdrawn from the mouse orbital sinus. Red blood cells were isolated with Easy Lyse Solution (10 mL; Leinco Technologies, USA), and then stained with Foxp3-PE, CD4-FITC, IL17A-APC, IFN-γ-Pacific Blue, IL4-PE, CD3-FITC and CD8-Pacific Blue (BioLegend, CA, USA). For graft analysis, the graft samples were homogenised and digested with collagenase (0.125%; Sigma, Missouri, USA) in a shaking water bath at 37 °C for 30 min. After centrifugation (800×g, 3 min) and resuspension, the cells were stained with the following monoclonal antibodies: CD11b-Pacific Blue, CD11c-PE/Cy7, Foxp3-PE, F4/80-PE, CD206-FITC CD4-FITC, IL17A-APC, IFN-γ-Pacific Blue, IL4-PE, CD3-FITC, and CD8-Pacific Blue (BioLegend, CA, USA). An irrelevant control monoclonal antibody was included for each fluorochrome. Finally, the cells were analysed using a BD LSR-II flow cytometer (Becton Dickinson, CA, USA). Cell Quest software (Becton Dickinson, CA, USA) was employed for data acquisition and analysis. A gate was set to exclude 99.9% of the isotype control-labelled cells.
3
Multicolor Flow Cytometry Panel
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Abs against following antigens: CD3-FITC, CD3-APC, CD25-FITC, CD4-PE, CD4-PECy7, CD8-PE, B220-PE, NK-1.1-PE, CD69-APC, FOXP3-PE, IFN-γ-APC, Isotype-matched control Ab, Annexin V-FITC, 7AAD, Purified anti-CD25 mAb (PC61) were purchased from BioLegend (San Diego, CA). The murine recombinant cytokines (IL-2, IL-12) were from Peprotech (Rocky Hill, NJ).
4
Multi-Color Flow Cytometry Immunophenotyping
5
Comprehensive Lung Cell Immunophenotyping
6
Antibody Acquisition for Immune Profiling
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Antibodies to phosphoAkt, phosphoSrc, phosphoSTAT3, and β-catenin were purchased from Cell Signaling Technology (Beverly, MA, USA). Antibodies to MART1, MelanA, CD8, and actin were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibodies to CD4, CD11b, CD25, NK1.1, IFN-γ and Ad5 were purchased from Novus Biologicals (Littleton, CO, USA). Antibody to CD11b+c was purchased from Thermo Fisher Scientific (Waltham, MA, USA). Antibodies to CD3-FITC, CD4-PE/Cy7, CD25-APC, and CD11c were purchased from Abcam (Cambridge, UK). Antibodies to CD122-PE/Cy7, CD8a-APC, FoxP3-PE, and NK1.1-APC were purchased from Biolegend (San Diego, CA, USA). All other chemicals were purchased from Sigma-Aldrich (St Louis, MO, USA).
7
Phenotypic Analysis of Murine Regulatory T Cells
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Half a million splenocytes were stained in each FACS tube. First, cells were washed with staining buffer (Biolegend, San Diego, CA, USA), and centrifuged at 800 g for 5 min at 4 °C. They were then stained with fluorochrome-conjugated antibodies after blocking of Fc receptors (CD16/32, Biolegend, San Diego, CA, USA) for 5 minutes. The following antibodies were used: FoxP3-PE, CD3-PE/Cy7, CD25-APC, CD4-PB, CD8a-FITC and CD3-PE-Cy7, all from Biolegend (San Diego, CA, USA). The cells were stained 30 min on ice in the dark with antibodies binding extracellular targets. Cells were then washed to remove unbound antibodies, incubated with Fixation/Permeabilization buffers (eBioscience, San Diego, CA, USA) for 30 min on ice and blocked with CD16/32 prior incubation with anti-FoxP3 for 30 min. Cells were resuspended in PBS buffer containing 1% BSA and 0.5 mM EDTA. Cells were run on Gallios flow cytometer (Beckman Coulter, High Wycombe, UK) and analyzed with FlowJo software V.10 (Tree Star, Inc. Ashland, OR, USA).
8
Regulatory T Cell Analysis in Lymph Nodes
9
Analyzing Human Regulatory T Cells
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Peripheral blood mononuclear cells (PBMCs) were isolated from 20 to 25 ml of peripheral blood samples via Ficoll density gradient centrifugation (Amersham Biosciences, Uppsala, Sweden). According to the recommendation of the international working group of the experts in flow cytometry [7 (link)], CD3, CD4, CD25, CD127, and FoxP3 markers were used to analyze human Treg cells using a gating strategy illustrated in Supplementary Figure 1. Specifically, CD3-FITC, CD45-PerCP, CD127-APC (Miltenyi Biotec, Bergisch Gladbach, Germany), CD4-Alexa Fluor® 700, (EXBIO a.s., Prague, Czech Republic), Foxp3-PE, and CD25-Brilliant Violet (BioLegend, San Diego, USA) antibodies (Abs) were used. For intracellular staining, we used Foxp3-Fix/Perm and Foxp3-PErm Buffers (BioLegend, San Diego, USA). The flow cytometric data were collected on a LSR II flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA) and analyzed using Summit 4.3 software (Dako, Glostrup, Denmark).
10
Comprehensive Treg Phenotyping by Flow Cytometry
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