Single-cell splenocyte suspensions were prepared by mincing the spleens in PBS containing 1% FBS (Gibco, Grand Island, NY, USA). Red blood cells were lysed using ACK lysis buffer. Approximately 106 splenocytes in 100 μl were stimulated with 50 ng/ml PMA (Sigma-Aldrich, St. Louis, MO, USA) and 1 μg/ml ionomycin (Sigma-Aldrich) in the presence of 10 μg/ml Brefeldin A (BD Biosciences, San Jose, CA, USA) for 4 h at 37 °C in 5% CO2. After 5 h incubation, the cells were surface stained with FITC anti-mouse CD4 antibody (BD Bioscience PharMingen, San Jose, CA, USA) at 4 °C for 30 min in the dark. Subsequently, the cells were fixed and permeabilized with Cytofix/Cytoperm buffer (BD Biosciences) and then intracellularly stained with PerCP-Cy5.5 conjugated anti-mouse IL-9 (BD Bioscience PharMingen). All of the above procedures were performed on ice until the time of analysis. All of the stained cells were evaluated using a Flow Cytometer (Beckman Coulter, Brea, CA, USA) and data were analyzed with CXP software.
Fitc anti mouse cd4 antibody
Manufactured by BD
Sourced in United States
The FITC anti-mouse CD4 antibody is a fluorescently labeled antibody that specifically binds to the CD4 antigen expressed on the surface of mouse T helper cells. It is used for the identification and enumeration of CD4+ T cells in flow cytometry applications.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Cytofix cytoperm buffer, by BD (2 mentions)
Ionomycin, by Merck Group (1 mentions)
Phorbol myristate acetate (pma), by Merck Group (1 mentions)
Brefeldin a, by BD (1 mentions)
Fcs express 4, by De Novo Software (1 mentions)
Anti mouse foxp3 pe, by Thermo Fisher Scientific (1 mentions)
Facscanto 2, by BD (1 mentions)
2 protocols using fitc anti mouse cd4 antibody
1
Intracellular Cytokine Staining of Splenocytes
2
Flow Cytometry Analysis of Murine Immune Cells
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Peripheral blood and PBS containing 1% FBS (Gibco, Grand Island, NY, USA) were used to prepare a single cell suspension. Cells were stained with FITC anti-mouse CD4 antibody (BD Bioscience PharMingen, San Jose, CA, USA) in a dark environment at 4°C for 30 minutes, and the cells were fixed and permeabilized with Cytofix/Cytoperm buffer (BD Bioscience PharMingen). They were then stained with PE anti-mouse IL-9 or PE anti-mouse IL-17 antibody (BD Bioscience PharMingen).18 (link) To detect Tregs, cells were stained with FITC anti-mouse CD4 and APC anti-mouse CD25 (eBioscience, San Diego, CA USA), then incubated at 4°C in the dark for 20 minutes. After being washed twice, the cells were fixed, permeabilized, and stained with PE anti-mouse Foxp3 (eBioscience, USA). Flow cytometric analysis (FACSCanto II, BD Biosciences, USA) was performed, and FCS Express 4 software (De Novo Software, Glendale, CA, USA) was used for analysis.
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