Apc conjugated anti mouse cd25

Manufactured by BD

Sourced in United States

APC-conjugated anti-mouse CD25 is a fluorochrome-labeled antibody that binds to the CD25 surface antigen on mouse cells. CD25 is the alpha chain of the interleukin-2 receptor and is expressed on activated T cells, regulatory T cells, and other immune cell types. This product can be used for the identification and analysis of CD25-expressing cells in mouse samples using flow cytometry.

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CD25-APC (96 citations) Anti-CD25-APC (52 citations) APC-conjugated anti-CD25 (12 citations) APC-conjugated CD25 (4 citations) Anti-mouse CD25 (3 citations) Mouse anti (2 citations)

6 protocols using apc conjugated anti mouse cd25

Immunophenotyping of T Cell Subsets

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Single cell suspensions were obtained from the spleens of NZB/W F1 mice at autopsy (at 43–44 weeks of age). The splenocytes were stained with PerCP-Cy5.5-conjugated anti-mouse CD3e (PerCP-cy5.5-CD3e, eBioscience, San Diego, CA, USA), fluorescein isothiocyanate (FITC)-conjugated anti-mouse CD4 (FITC-CD4, BD Biosciences, San Jose, CA, USA), PE-cyanine7-conjugated anti-mouse CD8a (eBioscience), allo-phycocyanin (APC)-conjugated anti-mouse CD25 (BD Biosciences) and PE-conjugated anti-mouse CD138 (BD Biosciences).
T cell profile was analyzed as described previously^{2 (link)}; briefly, we examined proportions of Th1 cells (CD4⁺CD25⁺T-bet⁺), Th2 cells (CD4⁺CD25⁺GATA-3⁺), Th17 cells (CD4⁺CD25⁺ROR-γt⁺), and Treg cells (CD4⁺CD25⁺Foxp3⁺). To analyze T helper subsets, the splenocytes were stained with antibodies to CD4 and CD25 (FITC-conjugated anti-mouse CD4 and APC-conjugated anti-mouse CD25, BD Biosciences). Cells were fixed and permeabilized prior to staining with T-bet, GATA-3, ROR-γt and Foxp3 antibodies (PE-, BD Biosciences).

Choi E.W., Song J.W., Ha N., Choi Y.I, & Kim S. (2018). CKD-506, a novel HDAC6-selective inhibitor, improves renal outcomes and survival in a mouse model of systemic lupus erythematosus. Scientific Reports, 8, 17297.

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Multiparametric Analysis of Immune Cell Subsets

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For Treg detection, cells were first stained with FITC-conjugated anti-mouse CD45 (BD, United States), PE-CY-7-conjugated anti-mouse CD4 (BD, United States), and APC-conjugated anti-mouse CD25 (BD, United States). For Th17 cell detection, cells were stimulated with Cell Stimulation Cocktail in culture medium for 4 h before surface staining using FITC-conjugated anti-mouse CD45 and PE-CY-7-conjugated anti-mouse CD4. After surface staining, cells were fixed and permeabilized with fixation/permeabilization buffer, followed by staining with PE-conjugated anti-mouse Foxp3 (eBIOSCIENCE, United States) for Tregs and PE-conjugated anti-mouse IL-17A (BD, United States) for Th17 cells. For dendritic cells detection, cells were stained with FITC-conjugated anti-mouse CD11c (BD, United States). Cells were analyzed on a FACS flow cytometer (CytoFLEX, Beckman, United States and BD Biosciences, United States).

Wang J., Liu T., Chen X., Jin Q., Chen Y., Zhang L., Han Z., Chen D., Li Y., Lv Q, & Xie M. (2021). Bazedoxifene Regulates Th17 Immune Response to Ameliorate Experimental Autoimmune myocarditis via Inhibition of STAT3 Activation. Frontiers in Pharmacology, 11, 613160.

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Multiparametric Flow Cytometry Analysis

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Isolated splenocytes were washed and resuspended in PBS. Cells were placed on ice and labeled with fluorescein isothiocyanate (FITC)-conjugated anti-mouse CD4⁺ (BD Pharmingen, CA), allophycocyanin (APC)-conjugated anti-mouse CD25 (BD Pharmingen), phycoerythrin (PE) conjugated anti-mouse CD73 (BD Pharmingen) and PE-Cy7-conjugated anti-mouse CD39 (eBiosciences, CA). Intracellular staining of splenocytes was also performed after surface staining in some experiments. For intracellular staining, cultured cells were collected and stimulated with leukocyte activation cocktail (PMA/ionomycin), with BD GolgiPlug (BD Biosciences) according to the manufacturer's protocol, for 4 h. Cells were incubated with FcBlock, stained with anti-CD4 (BD Biosciences), permeabilized, and stained with anti–IL-17A (eBiosciences), or anti–IFN-γ (eBiosciences), using a Cytofix/Cytoperm kit (BD Biosciences). Cells were also stained with their respective isotype controls as recommended in the manufacturer's protocols for adjusting gates. Samples were assayed with a BD FACSCalibur flow cytometer, and data were analyzed with FlowJo (TreeStar) software.

Alam M.S., Kuo J.L., Ernst P.B., Derr-Castillo V., Pereira M., Gaines D., Costales M., Bigley E, & Williams K. (2014). Ecto-5′-Nucleotidase (CD73) Regulates Host Inflammatory Responses and Exacerbates Murine Salmonellosis. Scientific Reports, 4, 4486.

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Murine Immune Cell Activation and Phenotyping

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All following conjugated Abs are from BD:APC conjugated anti–mouse CD25, -CD4, -CD45.1, -CD11c, -IL-12p70; Alexa Fluor 700–conjugated anti-CD3, -CD4, and -CD11c; PE-conjugated anti-CD3, -CD19, and -CD49b; FITC-conjugated anti-CD3, -CD19, -CD49b, and isotype control; biotin anti-CD4, -CD8, -DX5, -B220, -CD3, -CD11b, -Ly-6G, and -Ter119; and purified anti-CD16/CD32 (2.4G2). CD11c and streptavidin beads (SA) from Miltenyi Biotec; CFSE, live dead fixable aqua, CL075, and LPS from Invitrogen; ATRA from Sigma-Aldrich; hTGF-β1, anti–mouse TGF-β (1D11), anti-CTLA4, and Ig isotype control from R&D Systems.

Sela U., Park C.G., Park A., Olds P., Wang S., Steinman R.M, & Fischetti V.A. (2016). Dendritic Cells Induce a Subpopulation of IL-12Rβ2-Expressing Treg that Specifically Consumes IL-12 to Control Th1 Responses. PLoS ONE, 11(1), e0146412.

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Murine T Cell Phenotyping by Flow Cytometry

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Mesenteric lymph nodes isolated from each group of mice and CD4⁺ T cells cocultured with LPS- or WIKIM30-treated BMDCs were labeled with fluorescein isothiocyanate-conjugated anti-mouse CD4, allophycocyanin (APC)-conjugated anti-mouse CD25, peridinin chlorophyll-Cy5.5-conjugated anti-mouse T cell receptor β (BD Biosciences), or phycoerythrin (PE)-conjugated anti-mouse Forkhead box (Fox)p3 (eBioscience, San Diego, CA, USA) mAb. Flow cytometry was performed on a FACSCanto II system (BD Biosciences), and data were analyzed using FlowJo software (Tree Star Inc., Ashland, OR, USA).

Kwon M.S., Lim S.K., Jang J.Y., Lee J., Park H.K., Kim N., Yun M., Shin M.Y., Jo H.E., Oh Y.J., Roh S.W, & Choi H.J. (2018). Lactobacillus sakei WIKIM30 Ameliorates Atopic Dermatitis-Like Skin Lesions by Inducing Regulatory T Cells and Altering Gut Microbiota Structure in Mice. Frontiers in Immunology, 9, 1905.

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Flow Cytometric Analysis of Lymphocyte Subsets

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Lymphocyte cell suspension at a concentration of 2 × 10⁶ cells/mL (960 μL) and 40 μL of test agents were added to 24-well plates. Fresh RPMI 1640 medium containing 200 µg/mL FVPB2 were added into each well of 24-well microplate. After incubation for 48 h, lymphocytes were centrifuged, and washed twice with PBS (containing 1% BSA), and then labeled with different mAbs in PBS (containing 1% BSA) in the dark for 30 min at room temperature. After washing twice with PBS (containing 1% BSA), cells were resuspended in 0.3 mL of PBS, and sorted using BD AccuriC6 flow cytometery(BD FACSCalibur TM, Becton Dickinson, USA). FlowJo software (TreeStar, Ashland, OR) was used to analyze the percentage of various lymphocyte subpopulations. PE-conjugated anti-mouse CD19 conjugate, APC-conjugated anti-mouse CD25 and isotype control antibodies were from BD Biosciences (CA, USA).

Wang W.H., Zhang J.S., Feng T., Deng J., Lin C.C., Fan H., Yu W.J., Bao H.Y, & Jia W. (2018). Structural elucidation of a polysaccharide from Flammulina velutipes and its immunomodulation activities on mouse B lymphocytes. Scientific Reports, 8, 3120.

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