Figure 1.
Characterization of Fucci-expressing NMuMG cells.
(a–f) Flow cytometry analysis of Ki-67- and DAPI-stained NMuMG/Fucci cells. Fucci signals (mKO2 vs. mAG) of exponentially growing cells (a) and cells at confluency (d). Cells collected in gate #1 were found to be diploid (2 C) and Ki-67 negative, and thus were assigned to quiescent G0 phase. (g) Fluorescence images of NMuMG/Fucci cells, after a “wound” (scratch) was introduced into a confluent monolayer. Scale bar, 10 µm. (h, i) Time-lapse imaging (h) and fluorescence intensity (i) of mKO2-hCdt1(30/120) (red line) and mAG-hGem(1/110) (green line) in the cell indicated by the white arrowhead in (g).
Figure 2.
Quiescence-associated cell-cycle dynamics and Fucci signals.
(a, b) mKO2-hCdt1(30/120) and mAG-hGem(1/110) fluorescent probes label individual G1 phase nuclei (mKO2-positive) and S/G2/M phase nuclei (mAG-positive). (a) Cells in G1 phase during continuous cell division (cycling G1 cells) go into S/G2/M phase (gates #4 and #5) and express mAG before reaching mKO2 level comparable to those of non-dividing cells. (b) mKO2-positive cells can be subdivided into cycling G1 (gates #3 and #2) and quiescent G0 (gate #1). Accumulation of mKO2-hCdt1(30/120) continues after cell-cycle exit; subsequently, quiescent G0 cells exhibit high levels of mKO2-signal. (c) Schematized 5 populations gated according to Fucci signals. Note that cells in G0 exhibit the highest level of mKO2 signal (mKO2++ cells); cells in G1 exhibit lower mKO2 levels than cells in G0 (mKO2+ cells).
Figure 3.
Flow cytometric profiles of Fucci signals in immune cells from various organs of #639/#474 mice.
Single-cell suspensions from spleen, lymph node, Peyer’s patch, thymus, and bone marrow of #639/#474 mice were prepared and subjected to flow-cytometric analysis in order to analyze the levels of mAG and mKO2 signals. Data are representative of three individual experiments. Numbers in dot plots indicate mean ± S.D. of gated cells.
Figure 4.
Stimulation-induced changes in Fucci signals in various immune cell subsets isolated from #639/#474 mice.
(a) Splenocytes from #639/#474 mice were stained with APC-anti-CD3 mAb or APC-anti-CD19 mAb. Using flow cytometry, non-stimulated T and B cells were collected as CD3+ and CD19+ fractions, respectively. The cells were fixed and stained with Alexa 647-conjugated anti-Ki-67 mAb (solid line) and Alexa 647-conjugated control mAb (dotted line). The signals of Alexa 647 of cells in gate #1 and gate #5 are shown. (b) Cultured splenocytes were stimulated for 2 days with ConA and IL-2 or LPS, and T cells and B cells were gated as CD3+ and CD19+ cells, respectively. Data are representative of three individual experiments. Numbers in dot plots and histograms indicate mean ± S.D. of gated cells. (c) Sorted mKO2++ T cells (CD5+, gate #1) or B cells (CD19+, gate #1) were cultured with ConA or LPS, and Fucci-signals were analyzed at indicated time points. Data are representative of two individual experiments.
Figure 5.
Visualization of Fucci-signals of lymph node cells in #639/#474 mice.
(a–c) An inguinal LN was dissected and maintained at 37°C under superfused medium bubbled with 95% O2/5% CO2 and observed by 2-photon microscopy. Zoomed in on sequentially from (a) to (c). Orange squares in (a) and (b) are zoomed up in (b) and (c), respectively. (d-f) A popliteal LN 3 days post-innoculation of CFA emulsion with TNP (17)-KLH in the rear footpad. The LN was dissected and maintained at 37°C under superfused medium bubbled with 95% O2/5% CO2 and observed for 30 minutes. Zoomed in on sequentially from (d) to (f). Scale bars, 50 µm.
Figure 6.
Flow cytometric profiles of Fucci signals of differentiating T cell subsets in thymus of #639/#474 mice.
Thymocytes were stained with fluorochrome-conjugated anti-CD3, anti-CD4 and anti-CD8 mAbs, and subjected to flow cytometry. Total thymocytes were divided into CD4−CD8− cells (DN), CD4−CD8+CD3low cells (DN to DP transition), CD4+CD8+ cells (DP), CD4−CD8+CD3high cells (CD8 SP) cells and CD4+CD8− cells (CD4 SP), and levels of mAG and mKO2 signals in each subset are shown. Data are representative of three individual experiments. Numbers in dot plots indicate mean ± S.D. of gated cells.
Figure 7.
Flow cytometric profiles of Fucci signals of differentiating B cell subsets in bone marrow of #639/#474 mice.
(a) BM cells were stained with PE-Cy7-conjugated anti-B220 mAb followd by Alexa 647-conjugated anti-Ki-67 or contorol mAbs. B220+ cells were divided into 5 populations based on the Fucci-signals (Left). Signals of Alexa 647-conjugated anti-Ki-67 mAb (solid line with the same color) are shown (Right). (b) BM cells were stained with fluorochrome-conjuagated anti-B220, anti-IgM, and anti-CD43 mAbs for flow cytometry. B220+ cells were divided into pro-B cells as B220lowIgM−CD43+, pre-B cells as B220lowIgM−CD43− cells, immature B cells as IgM+B220low cells and circulating B cells as IgM+B220high cells. Levels of mAG and mKO2 signals in each subset are shown. Data are representative of three individual experiments. Numbers in dot plots indicate mean ± S.D. of gated cells.