Growth properties of purified N27-A and unpurified N27 cells.

<p><b>(A-F):</b> Both cell types were plated at 20,000 cells in each well of 6-well plates. Representative images were taken at Day 1, 3, and 5 for each cell type. Purified N27-A cells (<b>Images A-C</b>) grew more slowly than unpurified N27 cells (<b>Images D-F</b>). <b>(G)</b>: Growth charts of purified N27-A (red line) and unpurified N27 cells (green line) from Day 0 to Day 7. Data present the average cell number from two wells of purified and unpurified cells in three experiments (n = 6, each cell type). Bar, 20 μm for <b>A-F</b>.</p

Process of N27 cells clonal purification.

<p><b>(A):</b> Phase contrast image of unpurified N27 cells grown at low density. <b>(B):</b> Immunostaining for TH with green fluorescence in unpurified N27 cells. Shown is a cluster of cells exhibiting bright TH-positive staining. Most cells shown in phase contrast have no TH-immunoreactivity. <b>(C):</b> Schematic drawing of the clonal culture procedures for purifying N27-A cells. Cells were plated at low density to form individual colonies which were picked up and screened in 48- and 96-well plates. The positive clones were expanded in 6-well plates and 10-cm dishes. Bar, 20 μm for both <b>A</b> and <b>B</b>.</p

Western blots for TH and DAT in purified N27-A and unpurified N27 cells.

<p><b>(A)</b>: Representative images show TH, DAT and β-actin Western blots from purified and unpurified N27 cells. Strong TH bands were seen in purified N27-A cells, while much reduced TH protein levels were seen in unpurified N27 cells. There were moderate DAT protein levels in purified N27-A cells but only faint DAT bands in unpurified N27 cells. <b>(B):</b> Quantification of TH and DAT Western blots relative to β-actin bands. The TH and DAT levels in unpurified N27 cells were set at 100%. Results show that purified N27-A cells have four-fold higher TH and three-fold higher DAT protein levels compared to unpurified N27 cells. (n = 6, **<i>p</i><0.01)</p

Immunocytochemistry of purified N27-A and unpurified N27 cells for dopamine neuron markers TH and DAT.

<p>The N27 cells were cultured on 8-well chamber slides and immunostained for the dopamine neuron markers TH <b>(A-D)</b> and DAT <b>(E-H).</b> Other wells were double-stained for TH and the neuronal marker Tuj1 <b>(I-L).</b> To image every cell in each well, the nuclear marker DAPI was added to all wells. <b>(A-B):</b> The purified N27-A clone showed strong TH staining in all cells as demonstrated by dual-staining with TH and DAPI. <b>(C-D):</b> The unpurified N27 cell mixture revealed that only a small fraction of the DAPI-labeled cells were TH positive. <b>(E-F):</b> In the purified N27-A clone, all cells had moderate DAT staining as shown with DAT and DAPI double staining. <b>(G-H):</b> In the unpurified N27 cell mixture, very few cells were positive for DAT immunostaining. <b>(I-J)</b>: In the purified N27-A clone, all cells were double-positive for Tuj1 and TH. <b>(K-L)</b>: While there were few TH-positive cells in the unpurified N27 cell mixture, most cells were Tuj1 positive, demonstrating that the mixed cell population was neuronal. Bar, 50 μm for <b>A-L</b>.</p

Purified N27-A cells are more sensitive to 6-OHDA toxicity than unpurified N27 cells.

<p>Purified N27-A and unpurified N27 cells were cultured in 24-well plates (for trypan blue staining) and 96-well plates (for MTT assay). Two days after plating, cells were treated with 0–150 μM of 6-OHDA for 24 hr. The cell viability was measured by trypan blue staining <b>(A)</b> and MTT assay <b>(B)</b>. <b>(A):</b> Cell viability data from trypan blue staining showed that there were significantly fewer viable cells in purified N27-A cultures compared to unpurified N27 cultures. <b>(B)</b>: Cell viability results from the MTT assay also showed that purified N27-A cells had greater cell death than unpurified N27 cells after exposure to 6-OHDA. Reduced cell survival in both assays indicate that purified N27-A cells are more sensitive to 6-OHDA than unpurified N27 cells (n = 12 for <b>A</b>, n = 15 for <b>B</b>, **<i>p</i><0.01).</p

N27-A cells are more sensitive to MPP+ toxicity than unpurified N27 cells, and nomifensine can block MPP+ induced toxicity.

<p>Purified N27-A and unpurified N27 cells were cultured in 96-well plates at equal starting density. Two days after plating, cells were treated with 0–1000 μM of MPP+ for 24 hr. <b>(A):</b> Cell viability data from MTT assays showed that there was significantly greater cell death in N27-A cultures compared to that in N27 cultures after 100 μM to 1000 μM of MPP+ treatment (n = 12, *<i>p</i><0.05). <b>(B)</b>: After nomifensine pre-treatment, exposure to MPP+ did not significantly reduce cell viability in either N27-A or N27 cells, even when MPP+ reached 1000 μM (n = 12, <i>p</i>>0.3).</p

Dopamine neuron transcription factors (Nurr1, En1, FoxA2 and Pitx3) and VMAT2 and DβH in purified N27-A and unpurified N27 cell cultures.

<p>N27 cells were cultured on 8-well chamber slides and immunostained with the dopamine neuron transcription factors Nurr1 <b>(A-D)</b>, En1 <b>(E-H)</b>, FoxA2 <b>(I-L)</b> and Pitx3 <b>(M-Q),</b> as well as VMAT2 (<b>R-U</b>) and DβH (<b>V-Y</b>). Cells were also stained for the nuclear marker DAPI. <b>(A-B):</b> In N27-A clone, nearly all cells had strong Nurr1 staining as shown by Nurr1 and DAPI double staining. <b>(C-D)</b>: In unpurified N27 cells, only a small fraction of cells expressed Nurr1. <b>(E-F):</b> In N27-A cells, most cells had strong En1 staining. <b>(G-H)</b>: In unpurified N27 cells, nearly all cells were negative for En1 staining. <b>(I-J):</b> In N27-A clone, cells were moderately stained for FoxA2. <b>(K-L)</b>: In unpurified N27 cells, nearly all cells were FoxA2 negative. <b>(M-Q)</b>: In N27-A clone, cells were faintly stained for Pitx3. In unpurified N27 cells, no cells expressed Pitx3. (R-U): In N27-A clone, most cells were positive for VMAT2. In unpurified N27 cells, only a few cells were VMAT2-positive. (V-Y): In both N27-A and N27 clones, no cells were DβH-positive. Bar, 50 μm for <b>A-Y</b>.</p

Dopamine release from N27-A and N27 cells.

<p>N27-A and N27 cells were cultured in 12-well plates and treated with or without 10 μM nomifensine for 24 hr. Under basal conditions, N27-A cells had about 3-fold of higher dopamine concentrations in the medium than N27 cells (n = 4, ^#<i>p</i><0.01). In N27-A cells, dopamine concentrations were significantly increased when cells were treated with nomifensine, potassium depolarization, or nomifensine plus potassium depolarization compared to basal conditions (n = 4, *<i>p</i><0.05, **<i>p</i><0.01). In unpurified N27 cells, there was only a slight increase in dopamine release under these three conditions (n = 4, <i>p</i>>0.1).</p