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Fig. 5. In vivo repopulation assay of the testis SP EGFP-positive in busulfan-treated recipient analysed 10 weeks after transplantation. (A) Ho fluorescence flow cytometric analyses of ₆-integrin-positive cell fraction from EGFP donor cells. Sorting gate of the testis SP is enclosed in box. (B) Detection of fluorescent seminiferous tubules in a recipient testis transplanted with the EGFP testis SP as determined by fluorescence microscopic analysis of whole testis. (C) Flow cytometric analysis of EGFP and Ho blue fluorescences of testicular single cell suspensions prepared from recipient testis transplanted with the EGFP testis SP and (D) Ho fluorescence flow cytometric (red/blue Ho fluorescence) analysis of EGFP-positive cells in recipient testis gated from Fig. 5C. (E) Green and red fluorescence analysis of testicular cell suspension from recipient testis in order to determine EGFP-positive cell number per recipient testis by flow cytometry using TruCount^TM (BD Biosciences) methodology (see Materials and methods). EGFP-positive cells (EGFP) and TruCount fluorescent beads (FB) are boxed. (F) Enhanced colonization of recipient testis by the transplanted testis SP (n=8 testis) compared with total population (n=8 testis). EGFP-positive cell number per recipient testis was normalized to 10⁵ cells injected. The values are mean±s.e.m. Difference between SP and total cells is significant (P<0.03). Results were obtained from three independent experiments.

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