Anti cd3 bv510

Bone marrow cells from one femur were isolated and erythrocytes were lysed using 0.83% ammonium chloride. Cells were stained with eBioscience™ Fixable Viability Dye eFluor™ 780 according to the manufacturer’s protocol (Invitrogen, ThermoFisher Scientific). Cells were extracellularly stained with anti-CD3-BV510 (Clone 17A2, Nordic BioSite AB, Täby, Sweden), anti-CD4-FITC (Clone RM4-5, Nordic BioSite AB) and anti-CD25-APC (Clone 3C7, BD, Franklin Lakes, NJ, USA). Cells were then fixed and permeabilized using the FoxP3 staining buffer kit (Invitrogen, Thermofisher Scientific) and intracellularly stained with anti-Foxp3-PE (Clone FJK-16s, ThermoFisher Scientific) according to the manufacturer’s instruction. Treg cells were defined as CD4+CD25+Foxp3+, and results are expressed as the frequency of live cells. Samples were run on BD FACSVerse (BD), and data were analyzed using FlowJo software (version 10.4.1).

Bone marrow cells from one femur and mesenteric lymph nodes cells were isolated and erythrocytes in bone marrow were lysed using 0.83% ammonium chloride. Cells were stained with eBioscience Fixable Viability Dye eFluor 780 according to the manufacturer’s protocol (Invitrogen, ThermoFisher Scientific). Cells were extracellularly stained with anti-CD3-BV510 (Clone 17A2, Nordic BioSite AB, Täby, Sweden), anti-CD4-FITC (Clone RM4-5, Nordic BioSite AB, Täby, Sweden), and anti-CD25-APC (Clone 3C7, BD, Franklin Lakes, NJ). Cells were fixed and permeabilized using the FoxP3 staining buffer kit (Invitrogen, Thermofisher Scientific) and intracellularly stained with anti-Foxp3-PE (Clone FJK-16s, ThermoFisher Scientific) according to manufacturer’s instruction. Treg cells were defined as CD4+CD25+Foxp3+, and results are expressed as frequency of live cells or number of cells. Samples were run on BD FACSVerse (BD, Franklin Lakes, NJ), and data were analyzed using FlowJo software (version 10.4.1).