Research Paper Volume 4, Issue 4 pp 279—289

Figure 1. Magnetic-activated cell sorting (MACS) significantly enriches for SSCs isolated from rapamycin- or control-treated mice. Male animals were chronically administered rapamycin or control vehicle for 2 weeks, followed by MACS selection for undifferentiated germ cells. (A) Schematic diagram depicting the experimental design. Isolated testes were enzymatically digested to single cell suspensions containing germ cells (multi-colored circles). Incubation with magnetic beads conjugated to antibodies that recognize SSC surface proteins THY1 and GFRA1 was followed by the passage of the samples through separation columns attached to a magnet (gray bars). THY1+ and GFRA1+ cells were ultimately flushed out for cell culture or RNA isolation. (B) Quantitative RT-PCR was performed on THY1+/GFRA1+ cells enriched by MACS from age-matched wild type (non-injected) mice. When compared to the endogenous control Gapdh (assigned a relative value of “1”), the fold-changes in expression of SSC markers Gfra1, Plzf, and Oct4 were all significantly elevated in the MACS-selected cells versus unsorted testicular cells (16-fold, 10-fold, and 8-fold, respectively). Data represent mean values +/− SEM from three biological replicates. Student's t-test was performed to assess significance between each SSC marker and the endogenous control; ***p<0.001.