Isolation and culture of neurons and astroglia from rat cortex
Primary cortical neurons were isolated from one-day-old Sprague-Dawley rat pups and cultured according to the published methods as described by Chandler et al. [14]. The cells were plated on poly-l-lysine-coated, six-well plates at the concentration of 2 × 106 cells per well in fresh cortical medium [Dulbecco's Modified Eagle's Medium (DMEM, from Invitrogen, CA, USA) supplemented with 10% horse serum (Sigma, MO, USA), 25 mM glucose, 10 mM HEPES (Sigma), 2 mM glutamine (BioSource International, CA, USA), 100 IU/ml penicillin, and 0.1 mg/ml streptomycine]. To obtain pure neuronal cell cultures, the medium was replaced with the cortical medium supplemented with 5 μM cytosine-β-arabinofuranoside (Arac, from Calbiochem, CA) after 3 days of incubation (37°C, 5% CO2). After 2 days, the neuronal culture was switched back to cortical medium without Arac. The experiments were performed on 6- to 7-day-old neuronal cells. To obtain primary cultures of cortical astroglia, the cortical cells from one-day-old Sprague-Dawley rat pups were cultured in DMEM/Ham's F12 medium (1:1), 10% fetal bovine serum (Biomeda, CA, USA), 100 IU/ml penicillin, and 0.1 mg/ml streptomycine [15]. The cells were plated on poly-d-lysine coated, 6-well plates at the concentration of 2 × 106 cells per well. Cells were grown for 8–10 days (37°C, 5% CO2) and culture medium was changed every 2 days. Twenty-four hours prior to treatment with palmitic acid, the medium was changed to cortical neuronal cell culture medium.
Isolation and culture of astroglia from rat cerebellum
The enzyme digestion and trituration techniques were performed on the cerebellum tissue from 7-day-old rat pups, as described previously [16]. Briefly, dissected cerebellar tissue was placed in a cerebellar buffer solution containing 136.89 mM NaCl, 5.36 mM KCl, 0.34 mM Na2HPO4, 0.44 mM KH2PO4, 5.55 mM dextrose, 20.02 mM Hepes, and 4.17 mM NaHCO3, PH 7.4. Cerebellar tissue was minced, transferred to 0.025% trypsin solution in cerebellar buffer, and incubated in water bath for 15 minutes at 37°C. 0.04% DNase I solution in cerebellar medium (DMEM supplemented with 10% horse serum, 25 mM KCl, 5 mg/ml insulin, 50 μM GABA, 100 IU/ml penicillin, and 0.1 mg/ml streptomycine) was then added to inactivate trypsin. After the supernatant was collected from the trituration steps, the cells were separated from the debris into 4% BSA solution in cerebellar buffer supplemented with 0.03% MgSO4. Finally, the cells were plated onto poly-l-lysine coated 6-well culture dishes at a density of 2.0 × 106 cells/ml in 3 ml of fresh cerebellar medium. After 2 days, the media was switched to DMEM/Ham's F12 medium (1:1) to obtain pure cultures of cerebellar astroglia. The astroglia cell cultures with approximately 95% purity were obtained after 8–10 days culture (37°C, 5% CO2). Twenty-four hours prior to treatment with palmitic acid, the medium was changed to cortical neuronal cell culture medium.
Western blot analyses
For Western blotting, cells were washed three times with ice-cold TBS (25 mM Tris, pH 8.0, 140 mM NaCl, and 5 mM KCl) and lysed for 20 min by scraping into ice-cold lysis buffer. To extract membrane protein BACE1, lysis buffer containing 1% (v/v) Nonidet P-40, 0.1% (w/v) SDS, 0.5% (w/v) deoxycholate, 50 mM Tris, pH 7.2, 150 mM NaCl, 1 mM Na3VO4 and 1 mM PMSF [all chemicals from Sigma] was used [17]. For phosphorylated tau, lysis buffer containing 1% (v/v) Triton, 0.1% (w/v) SDS, 0.5% (w/v) deoxycholate, 20 mM Tris, pH 7.4, 150 mM NaCl, 100 mM NaF, 1 mM Na3VO4, 1 mM EDTA, 1 mM EGTA, and 1 mM PMSF [all chemicals from Sigma] was used [18]. The total cell lysate was obtained by centrifugation at 12,000 rpm for 15 min at 4°C. The total protein concentration was measured by BCA protein assay kit from Pierce (Rockford, IL, USA). Equal amounts of total protein from each condition were run at 200 V on 10% SDS-PAGE gels (BioRad, CA, USA). The separated proteins were transferred to nitrocellulose membranes for 1 h at 100 V and incubated at 4°C overnight with the appropriate primary antibodies [1:1000 BACE1 (Chemicon, CA, USA), 1:200 AT8 (Pierce Biotechnology, IL, USA), 1:2000 actin (Sigma)]. Blots were washed three times in PBS-Tween (PBS-T) and incubated with appropriate HRP-linked secondary antibodies (Pierce Biotechnology, IL, USA) diluted in PBS-T for 1 h. After an additional three washes in PBS-T, blots were developed with the Pierce SuperSignal West Femto Maximum Sensitivity Substrate (Pierce Biotechnology) and imaged with the BioRad ChemiDoc.
Data Analyses
Data are shown as means ± S.D. for three independent experiments. Student's t-test was used to evaluate statistical significances between different treatment groups. Statistical significance was set at p < 0.05.