Minimum Essential Medium (MEM), Dulbecco's Modified Eagle Medium (DMEM), Fetal Bovine Serum (FBS) were from Invitrogen (Carlsbad, CA). Monochlorobimane (MCB), 2,7′-Dichlorofluorescein diacetate (DCF-DA), Horse serum (HS), Bovine serum albumin (BSA), N-acetylcysteine (NAC), rotenone, paraquat, 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT), acivicin and bestatin were from Sigma (St. Louis, MO). Annexin Cy 3 was from Abcam (Cambridge, MA) and 7-Amino- Actinomycin D (7AAD) from BD Pharmingen (San Diego, CA).
2.2. Cortical Neuron Culture
Primary cortical neuron cultures were prepared from embryonic day 17 rats (Sprague-Dawley) as described (Dutton 1990
; Ramachandran et al. 2003
). Neurons were suspended in MEM containing 10% horse serum. They were plated in 6 well culture plates (1.2 ×106
cells/well) previously coated with poly-D-lysine. On the following day, neurons were treated with mitotic inhibitors to prevent astrocyte contamination. Cells were fully differentiated by day 5 in-vitro
and ready to use for the experiments detailed below.
2.3. Astrocyte Cultures
Cortical Astrocytes were prepared from the cerebral hemispheres of 2-day-old Sprague Dawley rat neonates as described by McCarthy and de Vellis (1980)
. In brief, brain cortices were removed; tissue was disrupted, treated with 2.5% trypsin and DNase, filtered through 0.25 μm sieve and seeded in 75 cm2
plastic flasks at a density of 6 × 106
cells. The cultures were grown to confluency in DMEM with 10% fetal bovine serum (FBS) and antibiotics. The medium was changed every 3 days and cultures were split on day 7 into the desired plates (100 mm, 60 mm or 6-well). The astrocytes were grown to confluency and were identified as more than 95% pure by staining with glial fibrillary acidic protein. All animal protocols were approved by Institutional Animal Care and Use Committee. Handling and treatment of animals were carried out according to the National Institutes of Health guidelines.
Cortical astrocytes prepared from 2-day-old Sprague Dawley rat cortices were suspended in a culture medium consisting of Eagle's minimal essential medium (MEM) with 10% fetal bovine serum and glutamine (2 mM), and plated in 75 cm2 cell culture flasks. At confluency, cells were lightly trypsinized and replated onto cell culture inserts (Fisher-Costar Brand, Fisher Scientific, Pittsburgh, PA, USA) at 2.5 × 105 cells/well and were used 7 days after re-plating, at which time they formed a confluent layer across the surface of the insert. Primary cortical neuron cultures prepared from embryonic day 17 rats were suspended in glial conditioned medium (MEM containing 10% horse serum) and plated into 6-well culture plates (1.2 × 106 cells/ well) previously coated with poly D-lysine. On the following day, neurons were treated with mitotic inhibitors to prevent astrocyte contamination. On the third day in vitro, inserts containing confluent astrocyte cultures were placed in wells containing neurons.
2.5. Toxin Treatments
On day 5 in vitro, neuron cultures were exposed to various concentrations of rotenone (0.025μM-1μM) and paraquat (2μM-100μM) for 4 and 24 h. Confluent astrocyte cultures were treated with 0.1μM- 100μM rotenone and 0.05 - 2mM paraquat. Parallel controls were used for baseline measures within cultures.
2.6. DNA Measures
DNA was assayed using a kit from Trevigen (Trevingen Inc., Gaithersburg, MD, USA). Cells were washed with PBS and re-suspended in serum-free medium. To 150 μL cell suspension, 50 μL diluted Hoechst dye solution were added and incubated at 37°C for 1 h. The fluorescence was recorded at excitation 350 nm and emission 460 nm. DNA content of the sample was calculated from a standard curve which used DNA from salmon testes (Aldrich, Milwaukee, WI, USA).
2.7. Viability Measures
Trypan blue (TB) exclusion
Cells were cultured as stated above and treated in the presence or absence of rotenone or paraquat. Subsequently, they were harvested by light trypsinization and suspended in defined media. A suspension of 0.2 ml was added to 0.5 ml trypan blue (0.4%) and 0.3 ml Hanks' balanced salt solution (HBSS). The number of trypan blue negative cells was counted using a hemocytometer to determine viable cells.
Another measure of viability utilized the MTT kit. Cultures were removed from the incubator, washed and MTT was added in an amount equal to 10% of the culture medium volume. Cultures were then returned to the incubator for 2 h. After incubation, the resulting formazan crystals were dissolved by adding an amount of MTT solubilization solution equal to the original media volume. This was mixed until all crystals were dissolved. Absorbance intensity was then measured at 570 nm, with background measured at 690 nm.
2.8. Measurement of ROS using 2, 7′-dichlorofluoroscein diacetate (DCF-DA)
Generation of reactive oxygen species (ROS) was estimated with the fluorescent probe, DCF-DA as previously described by Jung et al. (2001)
. Cells were harvested and washed with PBS and re-suspended at 1 × 106
cells/ml. DCF-DA was added to a final concentration of 20 μM and incubated for 30 min at 37°C. Following incubation, the cells were washed with PBS and re-suspended in PBS. ROS generation was measured by fluorescence intensity (505 nm Ex, 535 nm Em) of 10,000 cells with a FACS.
2.9. Measurement of Reduced Glutathione
The fluorometric GSH assays were based on those published by Ju et al. (2000)
, which used a quantization approach described by Fernandez-Checa and Kaplowitz (1990)
. Confluent monolayers were washed with phosphate buffered saline (PBS), then scraped and incubated with 100 μM monochlorobimane (MCB) for 10 min at 37°C
. The cells were lysed with triton X-100 (0.02%) and centrifuged at 13,000 g. Fluorescence of the MCB–GSH complex in the supernatant fluid was measured (360 Ex, 460 Em) on a Perkin Elmer HTS 7000 Bioassay Reader (Perkin Elmer, Wellesley, MA, USA). GSH content was calculated with a standard curve prepared from GSH incubated with MCB and glutathione-S-transferase (0.1U). GSH was expressed as mmoles/mg DNA.
Flow cytometry was also used to measure the MCB fluorescence intensity in large population (10,000 cells). After rotenone and paraquat exposure, cells were labeled with 100μM of MCB for 30 min. cells were scraped, washed once, re-suspended in cold PBS with 1% BSA, and immediately analyzed using FACS.
2.10. Annexin Cy3 and 7 AAD assay
Annexin Cy3 binding was used as a measure of apoptotic cell death. Following treatment, cells (floating and adherent) were harvested and stained with Annexin Cy3. Annexin Cy3 was added to each tube and incubated at room temperature for 5 min in the dark. Cells were washed, resuspended and stained with 5μl of 7 AAD and incubated for 10 min in the dark before analysis. Annexin Cy3 was analyzed by FACS (Ex 543nm and Em 570nm). The 7- AAD fluorescence was detected in the far red range (650nm).
2.11. Statistical Analysis
Results are expressed as mean ± SEM. One-way analysis of variance (ANOVA) followed by Students Newman Kuel's test was used for comparisons between the control and treated groups. A value of P<0.05 was considered statistically significant.