Splenic CD4+ cells from approximately 28-day-old mice were obtained using the CD4+ T cell isolation kit (Miltenyi Biotec, 130-104-454) or by sorting with the BD FACSAria II cell sorter. CD4+ cells were processed using the Mouse Th1/Th2/Th17 (BD Biosciences, 560758) and Th17/Treg (BD Biosciences, 560767) phenotyping kits. Cells were cultured for 3–4 hours with or without 50 ng/mL PMA and 1 μg/mL ionomycin and then harvested for RNA-Seq or flow analysis. The following antibodies were used: PerCP-Cy5.5 anti–mouse CD4 (BD Biosciences, 550954), PE anti–mouse IL-17a (BD Biosciences, 559502), FITC anti–mouse IFN-γ (BD Biosciences, 554411), APC anti–mouse IL-4 (BD Biosciences, 554436), and Alexa Fluor 647 anti–mouse FOXP3 (BD Biosciences, 560402).
Percp cy5.5 anti mouse cd4
Manufactured by BD
Sourced in United States
PerCP-Cy5.5 anti-mouse CD4 is a fluorescently labeled antibody that binds to the CD4 surface antigen on mouse cells. It is designed for use in flow cytometry applications to detect and quantify CD4-positive cell populations.
Automatically generated - may contain errors
Lab products found in correlation
Pe anti mouse il 17a, by BD (1 mentions)
Apc anti mouse il 4, by BD (1 mentions)
Alexa fluor 647 anti mouse foxp3, by BD (1 mentions)
Facsaria 2 cell sorter, by BD (1 mentions)
Cd4 t cell isolation kit, by Miltenyi Biotec (1 mentions)
Fitc anti mouse ifn γ, by BD (1 mentions)
Csampler analysis software, by BD (1 mentions)
Il 17a, by BD (1 mentions)
Pe anti mouse cd8a, by BD (1 mentions)
Lsrfortessa flow cytometer, by BD (1 mentions)
4 protocols using percp cy5.5 anti mouse cd4
1
Th Cell Phenotyping in Murine Spleen
2
Murine Splenic Immune Cell Analysis
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Individual mouse spleens were reduced to homogeneous single cells using 70 m nylon cell strainers (Corning, Tewksbury, MA, USA). Red blood cells (RBC) were processed three times or fewer using RBC lysis solution (Gibco, Grand Island, NY, USA) to count single cells. Finally, the cells were stained with PerCP-Cy5.5 Anti-Mouse CD4 (BD Biosciences, CA, USA), PE-conjugated Th1 (IFN-γ), or PE-conjugated Th17 (IL-17A) (BioLegend, San Diego, CA, USA). The fluorescence intensity was measured using a BD Accuri™ C5 flow cytometer system, and the data were processed using BD CSampler analysis software to determine the proportions of CD4+ and gated IFN-+ or IL-17A+ populations.
3
Multiparameter Flow Cytometry of Immune Cells
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MLN cells (1 × 106 cells/mL) were stained with PE anti-mouse CD8a, Percpcy5.5 anti-mouse CD4, and FITC anti-mouse CD3e monoclonal antibodies (10 μL) (BD Biosciences) for 15 min at room temperature in the dark. Stained cells were washed twice with PBS and fixed in 300 μL of PBS. Spleen cells (1 × 106 cells/mL) were stained with FITC anti-mouse CD4, APC anti-mouse IL-17a, PE anti-mouse IFN-γ, Percpcy5.5 anti-mouse IL-4, Percpcy5.5 anti-mouse CD4, PE anti-mouse CD25, and APC anti-mouse FOXP3 monoclonal antibodies. Flow cytometry data were analyzed using Flow Jo v10 software.
4
CFSE-Based T Cell Proliferation Assay
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Splenocytes suspended in CM (107/mL) were labeled with 1.1 µL of 5mM CFSE solution. Next, the lymphocytes were incubated for 5 min at RT in the dark. Following labeling, the cells were washed two times with PBS with 5% FBS and one time in PBS with 1% FBS and then centrifuged at 300× g for 5 min at 20 °C. Then, the cells were plated on 96-multiwell plates at a concentration of 1 × 106 cells/well per 200 µL of CM and stimulated with ConA (1 µg/well) or with OVA (10 µg/well) and incubated at 37 °C with 5% CO2. Following 120 h of culture, the cells were collected and additionally stained with PerCP-Cy5.5 anti-mouse CD4 (550954; BD Biosciences, San Diego, CA, USA) and analyzed using the BD LSRFortessa flow cytometer. FlowJo TM LLC software version 10.7.1 (BD Biosciences, San Jose, CA, USA) was used for the analysis of the results.
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