Bv421 conjugated anti mouse cd25 antibodies

Total thymocytes were isolated from the thymus in HSC buffer (HBSS with Ca2⁺ and Mg2⁺, 5% fetal bovine serum, 2 mM EDTA). Red blood cells (RBC) were lysed using ACK lysing buffer (Lonza). 1 x 10⁶ live thymocytes were blocked with a rat anti-mouse CD16/32 antibody (BD Pharmingen) and stained with a combination of the antibodies including PE-Cy5 conjugated anti-mouse CD4 (clone: GK1.5, eBioscience), PE conjugated anti-mouse CD8 (clone: 53-6.7, eBioscience), APC conjugated anti-mouse CD44 (clone: IM7, eBioscience), BV421 conjugated anti-mouse CD25 antibodies (clone: PC61, BioLegend). FITC conjugated anti-mouse CD45.2 (clone: 104, eBioscience), and APC-eFluor780 conjugated anti-mouse CD45.1 (clone: A20, e eBioscience). All antibodies were diluted 1:400. Data were collected from at least 100,000 single cells by FACSCanto (BD Pharmingen) and analyzed by FlowJo (Tree Star, Inc) without knowledge of the genotype or treatment by a single observer (C-LL).

Total thymocytes were isolated from the thymus according to methods described elsewhere (16 (link)). A total of 1 × 10⁶ live thymocytes were blocked with a rat anti-mouse CD16/32 antibody (BD Pharmingene, San Diego, CA) and stained with a combination of the antibodies including PE-Cy5 conjugated anti-mouse CD4 (clone: GK1.5), PE conjugated anti-mouse CD8 (clone: 53–6.7), APC conjugated anti-mouse CD44 (clone: IM7), FITC conjugated anti-mouse CD45.2 (clone: 104), and APC-eFluor780 conjugated anti-mouse CD45.1 (clone: A20), all acquired from eBioscience^™ Inc. (San Diego, CA), as well as BV421 conjugated anti-mouse CD25 antibodies (clone: PC61; BioLegend^® Inc., San Diego, CA). All antibodies were diluted 1:400. Data were collected from at least 100,000 single cells using FACSCanto^™ (BD Pharmingen) and analyzed using FlowJo (Tree Star Inc., Ashland, OR) without knowledge of the genotype or treatment by a single observer (C-LL).