Figure 1.
Figure 1. Time-lapse visualization of fetal thymus recruitment of T-precursor cells. (A) Diagram of time-lapse visualization assay in culture. (B) Representative images at indicated time points of the edge of a drop of 1 × 105 fetal liver (FL) cells (shown at the left side of the panels) in the absence (top panels) or presence of dGuo-treated fetal thymus lobe (shown at the right side of the bottom panels). Height and width of each picture at original magnification is 0.96 mm and 1.28 mm, respectively. (C) Means and SEs of the numbers of cells that moved out of the cell spot (□) and that reached the fetal thymus lobe (▨) were determined in microscopically visualized field. Numbers of independent experiments are indicated. (D) dGuo-treated fetal thymus lobes seeded with FL cells as in panels B and C were further cultured for 18 days under conventional organ culture conditions. Cells were 3-colored stained for CD3, CD4, and CD8. Shown are representative results (n = 5) of CD3 profiles (solid lines) and control staining profiles (dotted lines) of the cells within indicated CD4/CD8 subpopulations (as shown in dot plots). (E) Indicated FL cell fractions (1 × 105) and total fetal blood (FB) cells (5 × 105) from E14.5 embryos were examined for fetal thymus recruitment by time-lapse visualization assay. Bars are shown as in panel C. (F) Indicated thymocyte subpopulations (1 × 105) were examined for fetal thymus recruitment. Bars are shown as in panel C. Asterisks denote significantly smaller numbers than the cell numbers attracted from DN1 E14.5 fetal thymocytes (*P < .05; **P < .01; ***P < .001). Significance was evaluated individually for shaded bars and open bars. (G) dGuo-treated fetal thymus lobes or fetal salivary gland lobes were examined for the attraction of E14.5 fetal thymocytes. Bars are shown as in panel C. Thymus lobes or salivary gland lobes were cultured further for 9 days under conventional organ culture conditions. Cells were analyzed by flow cytometry for forward scatter (FSC) and side scatter (SSC) and for CD3, CD4, and CD8, as in panel D. Shown are representative results of 4 independent experiments. Numbers in quadrants indicate the frequency of cells within that box.

Time-lapse visualization of fetal thymus recruitment of T-precursor cells. (A) Diagram of time-lapse visualization assay in culture. (B) Representative images at indicated time points of the edge of a drop of 1 × 105 fetal liver (FL) cells (shown at the left side of the panels) in the absence (top panels) or presence of dGuo-treated fetal thymus lobe (shown at the right side of the bottom panels). Height and width of each picture at original magnification is 0.96 mm and 1.28 mm, respectively. (C) Means and SEs of the numbers of cells that moved out of the cell spot (□) and that reached the fetal thymus lobe (▨) were determined in microscopically visualized field. Numbers of independent experiments are indicated. (D) dGuo-treated fetal thymus lobes seeded with FL cells as in panels B and C were further cultured for 18 days under conventional organ culture conditions. Cells were 3-colored stained for CD3, CD4, and CD8. Shown are representative results (n = 5) of CD3 profiles (solid lines) and control staining profiles (dotted lines) of the cells within indicated CD4/CD8 subpopulations (as shown in dot plots). (E) Indicated FL cell fractions (1 × 105) and total fetal blood (FB) cells (5 × 105) from E14.5 embryos were examined for fetal thymus recruitment by time-lapse visualization assay. Bars are shown as in panel C. (F) Indicated thymocyte subpopulations (1 × 105) were examined for fetal thymus recruitment. Bars are shown as in panel C. Asterisks denote significantly smaller numbers than the cell numbers attracted from DN1 E14.5 fetal thymocytes (*P < .05; **P < .01; ***P < .001). Significance was evaluated individually for shaded bars and open bars. (G) dGuo-treated fetal thymus lobes or fetal salivary gland lobes were examined for the attraction of E14.5 fetal thymocytes. Bars are shown as in panel C. Thymus lobes or salivary gland lobes were cultured further for 9 days under conventional organ culture conditions. Cells were analyzed by flow cytometry for forward scatter (FSC) and side scatter (SSC) and for CD3, CD4, and CD8, as in panel D. Shown are representative results of 4 independent experiments. Numbers in quadrants indicate the frequency of cells within that box.

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