Cell phenotype was assessed by flow cytometry (BD LSRFortessa; BD Biosciences) after incubation with the following fluorescein-labeled antibodies: CD45-allophycocyanin (APC)-eFluor780, CD3e-fluorescein isothiocyanate (FITC), CD4-APC, CD8a-phycoerythrin (PE)-cy7, CD25-PE-cy7, Foxp3-PE, IFN-γ-AlexaFluor488, IL-4-PE-cy7, CD107a-PE, granzymeB-peridinin chlorophyll (PerCP)-eFluor710, perforin-FITC, CD11c-PE-cy7, CD80-PerCP-eFluor710, CD83-FITC, B7-H4-PE, and CD31-PE. All antibodies were purchased from eBioscience (San Diego, CA, USA), except CD4-APC, CD8a-PE-cy7, and Foxp3-PE (BD Biosciences). Intracellular antigens were determined after incubation with ionomycin (500 ng/mL; Abcam, Cambridge, UK) and phorbol-12-myristate13-acetate (10 ng/mL; Sigma-Aldrich, St. Louis, MO, USA) for 1 h and monensin (2 μM; eBioscience) for an additional 4 h. Fixation and permeabilization were performed prior to antibody incubation, and data were analyzed using FlowJo software (TreeStar, Ashland, OR, USA).
Foxp3 pe
Manufactured by BD
Sourced in United States
FoxP3-PE is a fluorochrome-conjugated monoclonal antibody used for the detection and quantification of the transcription factor FoxP3 in flow cytometry applications. FoxP3 is a key regulator of the development and function of regulatory T cells.
Automatically generated - may contain errors
Lab products found in correlation
Cd25 apc, by BD (17 mentions)
Cd4 fitc, by BD (11 mentions)
Cd3 fitc, by BD (9 mentions)
Facscanto 2, by BD (8 mentions)
Ionomycin, by Merck Group (6 mentions)
Cd4 pe, by BD (5 mentions)
Cd25 pe cy7, by BD (5 mentions)
Cd25 bv421, by BD (5 mentions)
Foxp3 transcription factor staining buffer set, by Thermo Fisher Scientific (5 mentions)
Il 17 pe, by BD (5 mentions)
Cd3 percp, by BD (5 mentions)
Cd4 apc, by BD (5 mentions)
Cd25 fitc, by BD (5 mentions)
Cd8 apc, by BD (5 mentions)
Percp cy5.5 cd4, by BD (4 mentions)
Il 4 pe, by BD (4 mentions)
Ifn γ pe, by BD (4 mentions)
Cd4 percp, by BD (4 mentions)
Cd3 apc cy7, by BD (3 mentions)
Cd11c pe cy7, by BD (3 mentions)
53 protocols using foxp3 pe
1
Flow Cytometry Immunophenotyping Protocol
2
Immunological Analysis of T Cell Activation
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Human IgD was purchased from Abcam (Cambridge, MA, USA). Anti-CD3 and CD28 antibodies were provided by T&L Biological Technology (Beijing, China). A770041 was purchased from Axon Medchem (Groningen, Netherlands). rhTNFR:Fc fusion protein was purchased from Guojian Pharmaceutical Company (Shanghai, China). Anti-mouse IgD antibodies were purchased from eBioscience (San Diego, CA, USA). Anti-human CD69-PE-cy5, CD154-PE, CD4-FITC, CD25-APC, IL-4-APC, IFN-γ-PE-cy7, IL-17-PE, and FoxP3-PE, along with anti-mouse CD3e-PerCP-CyTM5.5, CD4-FITC, CD25-APC, CD154-PE, IFN-γ-PerCP-CyTM5.5, IL4-APC, IL-17-PE, and FoxP3-PE antibodies were provided by BD Pharmingen (San Diego, CA, USA). Anti-Lck, anti-phospho-ZAP70, anti-phospho-Lck, anti-ZAP70, and anti-β-actin were purchased from Affinity Biosciences, Sigma and Abcam.
3
Immunological Assay Protocol
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RPMI 1640 was from Gibco, Carlsbad, CA. A Total RNA Extraction Kit was obtained from Solarbio (Beijing, China). BPA and OVA were obtained from Sigma-Aldrich (St. Louis, MO). A PrimeScript RT kit was ordered from Takara (Dalian, China). The APC-Cy7-CD3, FITC-CD4, PE-IL-4, PE-Foxp3, APC-Helios, and CBA Flex Set were obtained from BD Biosciences (Franklin Lakes, NJ).
4
Investigating Inflammatory Cytokine Regulation
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BD750 (#S0981) was purchased from Selleck (Shanghai, China). Epirubicin (#HY-13624) was purchased from MCE (New Jersey, USA). PrimeScript RT Master Mix (#RR036A) and TB Green® Fast qPCR Mix (#RR430A) were purchased from Takara (Dalian, China). Antibodies against IL-10 (#bs-0698 R), TNF-α (#bs-10,802 R), IL-1β (#bs-0812 R), and FoxP3 (#bs-10,211 R) were purchased from Bioss (Beijing, China). Antibodies against Claudin 1 (#13050-1-AP), Occludin (#27260-1-AP), and GAPDH (#10494-1-AP) were purchased from Proteintech (Wuhan, China). HRP-conjugated AffiniPure goat anti-rabbit IgG (H + L) (#SA00001-2) and FITC-conjugated AffiniPure goat anti-rabbit IgG (H + L) (#SA00003-2) were purchased from Proteintech (Wuhan, China). FITC-CD4 antibody (#553046) and PE-FOXP3 (#562466) antibody were purchased from BD Pharmingen (New York, USA). A mouse IL-10 ELISA kit (#ZC-37962), mouse IL-1β ELISA kit (#ZC-37974), mouse TNF-α ELISA kit (#ZC-39024), and mouse CCL2 ELISA kit (#ZC-385882) were purchased from Zcibio (Shanghai, China).
5
Immunophenotyping of Splenic T Cells
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The expression markers on T cells from spleen were determined by flow cytometry using the following antibodies: Percp-CD3, APC-γδT, PE-IL-17, and PE-Foxp3 purchased from BD Pharmingen (USA) or eBioscience (USA). Cell surface staining was performed according to the standard procedures. For intracellular detection of cytokines, cells were stimulated with phorbol-myristate-acetate (25 ng/mL; Sigma-Aldrich) and ionomycin (1 ng/mL; Sigma-Aldrich) in the presence of GolgiPlug™ (BD Pharmingen) for 4 h at 37°C in 5% CO2. The cells were then washed and stained with fluorescent antibodies against CD3 at room temperature in the dark. After surface staining, cells were fixed/permeabilized in fixation/permeabilization solution (Cytofix/Cytoperm™; BD Pharmingen) according to the manufacturer's protocol, and stained with anti-IL-17 mAbs/anti-Foxp3-mAbs for 30 min at 4°C. Cells were then washed with Perm/Wash Buffer (BD Pharmingen) and resuspended in PBS +2% FBS for flow cytometry analysis. Flow cytometry was performed on a BD FACS Canto II (BD Biosciences, USA) and analyzed using FCS Express 4 software (De Novo Software, USA).
6
Multiparameter Immune Cell Analysis
7
Isolation and Analysis of Murine Splenic Tregs
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The spleen was removed aseptically and placed on a 200-mesh sieve moistened with PBS. Then the spleen samples were ground using a grinding rod. After the erythrocyte lysate was added, the suspension was centrifuged and the supernatant was discarded. The pellet was washed with PBS, and splenocytes were maintained in 1640 medium containing 10% fetal bovine serum, 1% 100 U/mL penicillin, 100 μg/mL streptomycin. The cells were counted, and the cell concentration was adjusted to 1.0 × 107/mL.
To detect Treg cells, 200 μL of the single cell suspension was stained with APC-CD4 (BD Biosciences, USA), and PE-Foxp3 (BD Biosciences) monoclonal antibodies (MAbs), incubated at room temperature in the dark for 30 min, and analyzed by fluorescence-activated cell sorter (FACS). The data were evaluated using FlowJo software (BD Biosciences).
To detect Treg cells, 200 μL of the single cell suspension was stained with APC-CD4 (BD Biosciences, USA), and PE-Foxp3 (BD Biosciences) monoclonal antibodies (MAbs), incubated at room temperature in the dark for 30 min, and analyzed by fluorescence-activated cell sorter (FACS). The data were evaluated using FlowJo software (BD Biosciences).
8
Treg and MDSC Immune Cell Analysis
9
Characterization of T-cell Populations
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The cells were stained for surface and intracellular/intranuclear, and analysis by FACS Canto flow cytometer (BD Biosciences). The following fluorochrome conjugated Abs: PerCP Cy5.5-CD45, APC Cy7-CD45, PerCP Cy5.5-CD4, PE-CD4, FITC-CD4, FITC-CD3, APC-CD3e, PerCP-CD3e, APC Cy7-CD3, PE-CD11b,PE Cy7-CD11b, FITC-MHC-II, PerCP Cy5.5-MHCII, PE-CD19, FITC-CD19, PE Cy7-CD19, APC-CD25, PE Cy7-CD25, PerCp Cy5.5-CD25, APC-CD138, FITC-CD40, PE-FoxP3, FITC-IL10 and anti-CD16/32 (Fc block) were purchased from BD Pharmingen (San Diego, CA), Biolegend (San Diego, CA), or eBiosciences (Thermo Fisher Scientific, MA, USA). Frequencies and numbers of populations in the meninges, lymph nodes and spleens of B6 and BTBR mice were gated based on FSC-A and SSC-A, followed by gating in single cells and finally gated on CD45+ cells. Acquired FCS files were analyzed by Flow Jo-V10. Treg cell population were identified based on CD25+FoxP3+ cell in CD3+CD4+ population. To observe the Treg cell population, CD4+ cells were analyzed for the expression of CD25 and FoxP3 and gated on the double positive population. Data were obtained from three to four independent experiments with 4–6 pairs of mice in each experiment. In each experiment, equal number (2/3 males and 2/3 females from each group) of male and female mice were used.
10
Multicolor flow cytometry analysis of T cell subsets
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To distinguish live and dead cells, PBMC were stained with live/dead fixable stain dye (Life technologies). After PBS washing, cells were incubated with FITC-CD3 (BD Biosciences), PerCP-Cy5.5-CD4 (BD Biosciences), BV421-CD25 (BD Biosciences), APC-CD127 (Biolegend), and PE-Cy7-CD45RA (BD Biosciences). Cells were then fixed and permeabilized with Foxp3/Transcription Factor Staining Buffer Set (eBioscience) and further stained with PE-Foxp3 (BD Biosciences). Cells were analyzed with a FACSCanto II flow cytometer (BD Biosciences), and data were processed with FlowJo software (Tree Star, OR, USA).
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