Cytoplasmic TCRβ and membrane CD3 expression on CD8β-depleted, TCR-negative thymocytes. Total thymocytes were depleted and sorted for CD8β⁻CD27⁻CD69⁻ cells as described in Materials and Methods. Three-color flow cytometric analysis was done after staining the sorted cells with FITC-conjugated MoAbs against TCRβ, TCRγδ, CD8 and with CD4-TRC (left panels) or after staining the sorted cells with FITC-conjugated MoAbs against TCRβ, TCRγδ and with CD8-TRC (right panels). Dot plots shown are electronically gated on FITC⁻ cells. Histograms shown are electronically gated on FITC⁻, TRC⁺ cells (left panels TCRβ⁻TCRγδ⁻CD8⁻β⁻; right panels TCRβ⁻TCRγδ⁻CD8⁺β⁻). (A) cytoplasmic anti-TCRβ-PE staining on TCRβ⁻TCRγδ⁻CD8⁻β⁻ thymocytes (left panel) and TCRβ⁻TCRγδ⁻CD8⁺β⁻ thymocytes (right panel). The quadrants were chosen in such a way that greater than 99% of the dots of cells stained with isotype-matched control antibodies fell in the lower left quadrant (negative for both PE and FITC). Numbers in the quadrants represent percentages of cells. (B) Membrane CD3-PE staining (bold line) on TCRβ⁻TCRγδ⁻CD8⁻β⁻thymocytes (left panel) and TCRβ⁻TCRγδ⁻CD8⁺β⁻thymocytes (right panel). The shaded area in the histogram represents staining with the negative control (irrelevant IgGs). A representative analysis out of three independent experiments is shown.