Isolation of thymocytes, whole blood and spleen cells

Prior to cell isolation animals were sedated by isoflurane. At first, blood was drawn retrobulbar, immediately transferred into an EDTA tube and stored on ice. Hereafter, mice were euthanized by neck fracture. Spleen and thymus were isolated, transferred into phosphate buffered saline and stored on ice. Blood was then treated by 1 µl/100 µl anti CD16/CD32 (Fc block). 100 µl blood was immediately transferred into an ice cooled FACS tube and incubated on ice for at least 20 minutes. The rest of the blood was transferred into a 15 ml tube for TREC analysis. Thymus cells were separated through a 40 μm cell strainer. Remnants, which mainly contain thymic stroma cells, were collected dry into a 1 ml tube and stored at −80 °C till further treatment. Thymic single cell suspension was then centrifuged (5 min, 500 x g, 4 °C) and resuspended in 1 ml MACS buffer (0,5 % BSA, 0,02 % NaN3, 2 mM EDTA in PBS, pH 7,4). Spleen cells were separated through a cell strainer according to thymic cells. After centrifugation (5 min, 500 x g, 4 °C) and removing supernatant, erythrocyte lysis was performed by resuspending cell pellet in 8 ml BD Pharm Lyse™ for 5 minutes at RT. Lysis was stopped by adding the same volume of PBS. Cells were then centrifuged (5 min, 500 x g, 4 °C), washed in 5 ml PBS, resuspended in 2 ml MACS buffer and stored on ice till further treatment.