Carl Zeiss Inc.). Positive staining and total cells (2,000 cells for each marker) were counted and analyzed as described in the immunocytochemistry method. Western Blot Cell extracts were obtained with RIPA buffer in the presence of protease inhibitors (sodium pyrophosphate, sodium fluoride, phenylmethanesulfonyl fluoride, pepstatin A, aprotinin, leupeptin, antipain, benzamidine - all from Sigma). Eighty micrograms of protein extract from MSC (non-induced, serum-free and 8 h chemically-induced groups), bone marrow mononuclear cells, NIH 3T3 (non-induced and chemically-induced) and adult rat brain cortex were electrophoretically separated in a 10% acrylamide gel and transferred onto a nitrocellulose membrane (GE HealthCare, England). The blot was probed for NeuN (Chemicon, 1:1000), NF-200 (Sigma, 1:500), b-tubulin III (Sigma, 1:1000), stripped, and probed for actin (Chemicon, 1:2000). Secondary antibodies were horseradish peroxidase conjugated (HRP, Santa Cruz Biotechnology, Santa Cruz, CA, USA) and detected with ECL reagents (GE HealthCare, England). Light- sensitive silver radiographic films (HyperFilm, GE HealthCare, Sweden) were exposed to ECL and developed. Flow Cytometry Non-induced, serum-free and chemically-induced MSC were evaluated for cell death during the induction protocol, using Annexin V and propidium iodide (PI) as markers for apoptosis or necrosis, respectively (Annexin V-FITC Apoptosis Detection Kit I; BD Biosciences, San Diego, CA, USA). The chemically-induced MSC group was divided into 4 subgroups: BME, where cells were treated only with BME for 24 h; 24 h BME treated cells induced with the DMSO/BHA mix for 4, 8 and 24 h. Adherent cells were trypsinized and collected in a tube, followed by centrifugation to remove trypsin and medium. Cells were then washed in PBS twice and resuspended in 100 mL of Binding Buffer (BD Biosciences). Subsequenlty, 5 mL of anti-Annexin V antibody and 10 mL of PI were added to each tube, and cells were incubated at room temperature for 15 minutes. MSC from the non-induced group were used to set the cytometer parameters: 1) without Annexin V and PI; 2) Annexin V only; 3) PI only. Cell counts were taken in a BD FACSCalibur (BD Biosciences) and analyzed with Cell Quest Pro software (BD Biosciences). The following parameters were used: FL1 for Annexin V, FL2 for PI, side scatter and forward scatter. The experiment was made in triplicates (n = 3) and 30,000 events were registered for each sample. Statistical analyses were performed using ANOVA with Newman-Keuls post-hoc test. Significance level was set at p,0.05. Homocysteine and cysteine quantification Non-induced and 8 h chemically-induced MSC cultures (n = 9, each group) were used for biochemical analysis. Cells were washed 3 times in PBS, and detached from the plate surface with cell scrapers in the presence of protease inhibitors (sodium pyrophos- phate, sodium fluoride, phenylmethanesulfonyl fluoride, pepstatin A, aprotinin, leupeptin, antipain, benzamidine - all from Sigma) diluted in PBS. Cells were collected in 2 mL cryovial tubes and submitted to five freeze and thaw cycles, by alternating between dry ice and water bath at 37uC. Cell lysates were stored at 280uC until chemical analysis. Intracellular Hcy and Cys were measured by high performance liquid chromathography with fluorimetric detection and isocratic elution. This methodology was adapted from Pfeiffer et al [33] and involves three steps, namely reduction of thiol groups using TCEP (tris(CarboxyEthyl)Phosphine), protein precipitation with trichlor- Induced MSC Are Not Neurons oacetic acid, and derivatization with SBD-F (7-fluorobenzene-2- oxy-1,3-diazolic-4-ammonium sulfate). The Shimadzu high per- formance liquid chromathography system had a SIL-10ADvp automatic sample injector and a RF-10AXL fluorescence detector. Chromatographic separation was performed using a C18 model Shim-pack CLC-ODS column (4.66150 mm, with 5.0 mm micro particles). The fluorescence was detected at a 385 nm excitation and at a 515 nm emission. Total Hcy and Cys levels were measured using a calibration curve of defined Hcy and Cys concentrations with cystamine as the internal standard normalized to the total protein content of the cell extract. Due to inter-assay variation, total Hcy and Cys levels were expressed as the ratio of each measure to the mean content of the control values. Statistical analyses were performed using t-Student test with significance level set at p,0.05. Electrophysiology In vitro patch clamp recordings were used to distinguish chemically-induced MSC from neurons by their electrophysiolog- ical features, such as action potential firing properties, presence of K+ and Na+ currents, and presence of voltage-gated channels. Neurons from primary culture of E17 cortex and standard cultured MSC were used as controls. The cell-attached voltage-clamp and whole-cell current-clamp or voltage-clamp configurations were performed in cells placed in a bath solution consisting of (in mM) 137 NaCl, 2.7 KCl, 1.36 NaH2PO4, 0.49 MgCl2 6H2O, 11.9 NaHCO3, 1.36 CaCl22 H2O, and 5.04 Glucose, and with a pH of 7.4 (Tyrode). Whole-cell pipette recordings were obtained from either MSC or neurons visually identified using a microscopy system (model Diaphot, Nikon). Patch electrodes (3–7 MV for whole cell or 8–15 MV for cell-attached) were pulled from 1.5 mm o.d. borosylicate glass capillary tubing using a micropipette puller. For whole-cell K+ current recordings, the intracellular solution contained (in mM) 1 CaCl2, 140 KCl, 1 MgCl2, 10 HEPES, 10 EGTA, 2 Na2ATP and 0.2 Na2GTP, and had a pH of 7.2. To study the K+ current, the holding membrane potential was 250 mV. The voltage-clamp protocol consisted of 100-ms depolarizing steps with 10-mV increments at 0.5 Hz, taking the membrane from 260 to +60 mV. For whole-cell Na+ current recordings, intracellular solution consisted of (in mM) 140 CsCl, 2 K-ATP, 4 MgCl2, 10 EGTA and 10 HEPES, had a pH of 7.3, and 300 Osm. Moreover, 4-aminopyridine (5 mM) and tetraethylamonium (10 mM) were added to the Tyrode extracellular solution to block K+ currents. The voltage-clamp protocol to study the Na+ current consisted of a 100-ms hyperpolarizing pulse from a holding potential of 280 to 2120 mV to remove channel inactivation, followed by 200-ms depolarizing steps with 10-mV increments at 0.5 Hz, bringing the membrane from 280 to +70 mV. Intracellular patch pipette solution for whole-cell current-clamp recordings contained (in mM) 120 K-gluconate, 10 KCl, 1 MgCl2, 0.025 CaCl2, 0.2 EGTA, 2 Na2-ATP, 0.2 Na2-GTP and 10 HEPES, had a pH of 7.2, and 295 Osm. In current-clamp, current pulses (50–1000-ms, 0.5 Hz) were given through the patch pipette to examine whether the cells were capable of producing action potentials. Single-channel currents were recorded using the cell-attached mode of the patch-clamp technique. Cell-attached patches were performed in the presence of high-KCl solution, both in the bath and pipette, which contained (in mM) 150 KCl, 1 MgCl2.6H2O, and 10 HEPES, and had a pH of 7.4. Patches were examined at a holding potential of +60 mV. After a patch was obtained, a stabilization period of ,5 minutes was allowed; patches that showed large fluctuation in channel activity over this period were discarded. Thereafter, channel activity was recorded continuously PLoS ONE | www.plosone.org 3 April 2009 | Volume 4 | Issue 4 | e5222