Single cell suspension of splenocytes, lymph node cells, or thymocytes were made, and these were stained with antibodies CD3-APC (OKT-3, BD Biosciences), CD4-V450 (RM4-5, eBioscience), CD8-V500 (RPA-T8, BD Biosciences company), Vβ8-PE (F23.1, BD Biosciences) KI-67-PerCp-Cy5.5 (BD56, BD biosciences), CD25-PerCp-Cy5.5 (PC61.5, Ebioscience), IFN-γ-FITC (XMG1.2, BD biosciences), CD44-APC (IM7, ebioscience), CD62L-FITC (MEL-14, BD biosciences) or FoxP3-eFluor450 (FJK-16s, ebioscience) and incubated for 30 min at 4°C. Cells were washed three times with PBS containing 2% FCS. Cells were acquired on the FACS Canto II (BD) and analyzed with FlowJo 7 (Tree Star). For cell activation experiments, splenocytes from transgenic mice or littermates were cultured (1 × 105 cells/well) for 24 h in the presence of 20 μg/ml mB29b, in which the last 4 h was in the presence of 1 μg/ml Brefeldin A.
Cd25 percp cy5 5 pc61
Manufactured by Thermo Fisher Scientific
CD25-PerCp-Cy5.5 (PC61.5) is a fluorescently labeled antibody that binds to the CD25 antigen. It is designed for use in flow cytometry applications.
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Lab products found in correlation
Cd8 v500 rpa t8, by BD (1 mentions)
Cd62l fitc mel 14, by BD (1 mentions)
Ifn γ fitc xmg1, by BD (1 mentions)
Cd44 apc im7, by Thermo Fisher Scientific (1 mentions)
Foxp3 efluor450 fjk 16s, by Thermo Fisher Scientific (1 mentions)
Facscanto 2, by BD (1 mentions)
Cd5 percp cy5.5 53 7 3, by Thermo Fisher Scientific (1 mentions)
Live dead fixable blue stain kit, by Thermo Fisher Scientific (1 mentions)
Lsrfortessa flow cytometer, by BD (1 mentions)
2 protocols using cd25 percp cy5 5 pc61
1
Phenotypic Analysis of Activated T Cells
2
Multiparametric Flow Cytometry of Thymocyte Subsets
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Flow cytometric analysis was performed as described recently [54] . Cell suspensions isolated from thymi were labeled directly with the following fluorochrome-conjugated antimouse antibodies purchased from either BioLegend, BD Biosciences, or eBioscience: CD4-BV605 (RM4-5), CD8α-APC (53-6.7), CD25-PerCP-Cy5.5 (PC61.5), and human CD2-BV421 (RPA-2.10). Detection of active caspase 3/7 was accomplished using CellEvent™ Caspase-3/7 Red Detection Reagent (ThermoFisher Scientific). Sorted CD4SP Rag1 GFP+ thymocytes were stained with CD5-PerCP-Cy5.5 (53-7.3 ) and Nur77-PE (12.14) antibody (both from eBiosciences). Exclusion of dead cells was done by using the LIVE/DEAD™ Fixable Blue stain kit (Invitrogen). Cells were acquired on LSR Fortessa™ flow cytometer (BD Biosciences), and data were analyzed with FlowJo software (BD Biosciences).
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