Purification of TCRαβ⁺ DN T cells via magnetic beads and flow cytometric sorting and TCR Vβ repertoire of purified DN T cells. (A) Purification of TCRαβ⁺ DN T cells using a Dynabeads-based depletion system to eliminate CD4⁺, CD8⁺, CD14⁺, CD19⁺, and CD56⁺ cells, as well as flow cytometric sorting with anti-TCRαβ, anti-CD4, and anti-CD8 mAbs. (B) Comparative analysis of the TCR Vβ repertoire of purified DN and CD4⁺ T cells. (C) Total RNA from sorted DN T cells was extracted, reverse transcribed, and amplified by reverse transcription-PCR (RT-PCR) using Vβ primers. Amplified cDNA was copied with a fluorescent Cβ primer in a run-off reaction and subjected to electrophoresis on an automated sequencer. The patterns obtained show the size in amino acids of the CDR3 region (x-axis) and the relative fluorescence intensity (y-axis) of in-frame Vβ-Cβ amplification products.