Splenocytes were stained with 10 mg/ml D b NP 366 or 40 mg/ml D b N3A 366 -PE tetramer in sort buffer (PBS with 0.1% BSA) for 60 mins at room temperature, washed and stained with 1 mg/ml anti-CD8-Allophycocyanin and 10 mg/ml of either anti-Vb 8.3 or anti-Vb9 for 30 mins on ice, washed twice with sort buffer. Single lymphocytes were isolated by sorting with a FACS Aria (BD Immunocytometry), into 80 wells of an empty 96 well twintec plate (Eppendorf). mRNA transcripts were reversed transcribed to cDNA, using a Sensiscript kit (Qiagen) according to manufacturers instructions, and the CDR3b region amplified by a nested hot start PCR using Vb primers [34] . Positive PCR products were purified using Qiagen PCR purification kit and sequenced.