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Most of the tomato-negative cells expressed α-smooth muscle actin (α-SMA), a peritubular myoid cell marker, but double-negative populations were also present. These findings suggest that vimentin lacks Sertoli cell-specificity and that α-SMA is not adequate to identify all of the contaminating cells. Upon FACS sorting; however, virtually 100% of the cells were tdTomato positive, expressed vimentin, but not α-SMA. Prepubertal mice yielded a higher number of Sertoli cells compared to adults, but both could be adequately sorted. In conclusi