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. 2009 Sep 17;106(40):17163–17168. doi: 10.1073/pnas.0905016106

Fig. 2.

Fig. 2.

Correlation of in vivo differentiation status of NSC/NPC and GFP fluorescence intensity in the NS-GFP-Tg brain. (A) High GFP expression correlates with high nestin expression in cells of NS-GFP-Tg embryonic forebrains. Coronal sections of forebrains of E14.5 NS-GFP-Tg mice were subjected to immunohistochemical analysis using anti-GFP (green) plus anti-type III β-tubulin (TuJ1; red, Top), or anti-GFP plus anti-nestin (red, Middle and Bottom). Images of the ventricular zone (VZ) cells in the Middle are shown at higher magnification in Bottom. (Scale bars: Top and Middle, 100 μm; Bottom, 10 μm). (B) Fractionation of GFP-expressing cells from E14.5 NS-GFP-Tg brain. Total brain cells from E14.5 NS-GFP-Tg mice were dissociated and fractionated by flow cytometry into 4 subpopulations, GFP−/+, GFP++, GFP+++ and GFP++++, based on GFP fluorescence intensity as indicated. Gray peak, WT C57BL/6 mice; black peak, NS-GFP-Tg mice. (C) Correlation of high GFP expression with immature cell properties. The fractionated brain cell subpopulations from B were fixed and analyzed by immunocytochemistry to detect expression of nestin (red, i–iv) or TuJ1 (red, v–viii) as indicated. Blue, nuclear marker TOTO-3. (Scale bars, 20 μm.) (D) Correlation of GFP expression with neurosphere-forming capacity in E14.5 NS-GFP-Tg brain tissues. Fractionated brain cell subpopulations from B were cultured under conditions allowing neurosphere formation. Data shown are the mean number ± SD. of neurospheres generated per 2,000 cells (n = 3 per group). (E) Fractionation of P3 GFP-expressing brain cells. Total brain cells from P3 NS-GFP-Tg mice were dissociated and fractionated by flow cytometry into 4 subpopulations, GFP−/+, GFP++, GFP+++ and GFP++++, based on GFP fluorescence intensity as indicated. Gray peak, WT C57BL/6 mice; black peak, NS-GFP-Tg mice. (F) Correlation of GFP expression with neurosphere-forming capacity in P3 NS-GFP-Tg brain tissues. The fractionated brain cell subpopulations in E were cultured to allow neurosphere formation as for D. Data shown are the mean number ± SD. of neurospheres generated per 2,000 cells (n = 3 per group).

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