Fig 1.
Process of N27 cells clonal purification.
(A): Phase contrast image of unpurified N27 cells grown at low density. (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. (C): 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 A and B.
Fig 2.
Growth properties of purified N27-A and unpurified N27 cells.
(A-F): 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 (Images A-C) grew more slowly than unpurified N27 cells (Images D-F). (G): 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 A-F.
Fig 3.
Immunocytochemistry of purified N27-A and unpurified N27 cells for dopamine neuron markers TH and DAT.
The N27 cells were cultured on 8-well chamber slides and immunostained for the dopamine neuron markers TH (A-D) and DAT (E-H). Other wells were double-stained for TH and the neuronal marker Tuj1 (I-L). To image every cell in each well, the nuclear marker DAPI was added to all wells. (A-B): The purified N27-A clone showed strong TH staining in all cells as demonstrated by dual-staining with TH and DAPI. (C-D): The unpurified N27 cell mixture revealed that only a small fraction of the DAPI-labeled cells were TH positive. (E-F): In the purified N27-A clone, all cells had moderate DAT staining as shown with DAT and DAPI double staining. (G-H): In the unpurified N27 cell mixture, very few cells were positive for DAT immunostaining. (I-J): In the purified N27-A clone, all cells were double-positive for Tuj1 and TH. (K-L): 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 A-L.
Fig 4.
Dopamine neuron transcription factors (Nurr1, En1, FoxA2 and Pitx3) and VMAT2 and DβH in purified N27-A and unpurified N27 cell cultures.
N27 cells were cultured on 8-well chamber slides and immunostained with the dopamine neuron transcription factors Nurr1 (A-D), En1 (E-H), FoxA2 (I-L) and Pitx3 (M-Q), as well as VMAT2 (R-U) and DβH (V-Y). Cells were also stained for the nuclear marker DAPI. (A-B): In N27-A clone, nearly all cells had strong Nurr1 staining as shown by Nurr1 and DAPI double staining. (C-D): In unpurified N27 cells, only a small fraction of cells expressed Nurr1. (E-F): In N27-A cells, most cells had strong En1 staining. (G-H): In unpurified N27 cells, nearly all cells were negative for En1 staining. (I-J): In N27-A clone, cells were moderately stained for FoxA2. (K-L): In unpurified N27 cells, nearly all cells were FoxA2 negative. (M-Q): 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 A-Y.
Fig 5.
Western blots for TH and DAT in purified N27-A and unpurified N27 cells.
(A): 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): 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, **p<0.01)
Fig 6.
Purified N27-A cells are more sensitive to 6-OHDA toxicity than unpurified N27 cells.
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 (A) and MTT assay (B). (A): 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): 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 A, n = 15 for B, **p<0.01).
Fig 7.
N27-A cells are more sensitive to MPP+ toxicity than unpurified N27 cells, and nomifensine can block MPP+ induced toxicity.
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. (A): 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, *p<0.05). (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, p>0.3).
Fig 8.
H2O2 toxicity is similar in purified and unpurified N27 cells.
Purified N27-A cells 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 0–200 μM of H2O2 for 24 hr. Cell viability was measured by trypan blue staining (A) and MTT assay (B). (A): Cell viability data from trypan blue staining showed similar numbers of viable cells in purified and unpurified N27 cells. (B): Cell viability results from the MTT assay revealed no significant differences in surviving cells from purified N27-A or unpurified N27 cells indicating that both cell sources had similar sensitivity to the non-specific toxicity of H2O2 (n = 12 for A, n = 15 for B, **p<0.01).
Fig 9.
Dopamine release from N27-A and N27 cells.
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, #p<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, *p<0.05, **p<0.01). In unpurified N27 cells, there was only a slight increase in dopamine release under these three conditions (n = 4, p>0.1).