FACS analysis of cells differentiating in the T-lymphopoiesis system. (A) Input cells: top panel is forward scatter (Fsc) versus side scatter (Ssc) profile showing the gate (G1) used for analysis of lymphocyte-like cells. The middle panel is a flow cytometric profile of surface CD2 and CD34 expression of MNCs used for flow cytometric purification. The R1 gate (1% to 2% MNCs) was established to sort CD34⁺CD2⁻ cells by comparing positive and negative stained controls. The lower panel is a representative flow cytometric analysis of CD34⁺CD2⁻ cells isolated by flow cytometry or magnetic bead separation before culture on thymic stroma. Quadrants were set using matched isotype controls, and comparable purification results were obtained in multiple independent experiments. (B) Differentiation of cells expressing surface CD2, CD3, CD4, and CD8 in human thymic stroma culture is not perturbed by exposure to rAAV. FACS analysis of CD34⁺CD2⁻ cells either mock-transduced (upper panel) or exposed to rAAV (lower panel) after 21 days in the T-lymphopoiesis system. Quadrants were established using matched isotype antibody controls and represent one of three comparisons. Minor cell populations expressing nonlymphoid phenotypes were detectable at early time points, but these were absent at later time points (data not shown).