Abeta 解聚实验方法.docx

C On-line MethodsPreparation of oligomer form of A642.• Synthetic A^42 were purchased from American Peptide (Sunnyvale, CA, USA) and prepared following the protocols described previously.1. The Ap42 peptide was dissolved in 191,1939393-hexafluoro-2-propanol (HFIP, Sigma) at 1 mg/ml concentration, and was aliquoted in Eppendorf tubes (100 yg/tube).2. The HFIP was allowed to evaporate in the fume hood and the resulting clear peptide film was dried under vacuum overnight and then pallets were kept at -20°C until use.3. For oligomerisation of Ap42 ( MW:4514.08), the dried pallet was re-suspended in DMEM or PBS to a final concentration of 100 |1M , followed by incubation at 4° C for 24 hours. The oligomer form of A^42 was checked by Wester n blot before use. (add 221.5 |1L DMEM)• The thioflavin T (ThT) fluorescence assay.1. To test the inhibitory effect of gastrodin on Afi aggregation, 1 |iM Ap42 (4. 514 |ig ) was incubated with a serial gradient of gastrodin (0 |1M, 0.156 |1M, 0.313 |1M, 0.625 |1M, 1.125 |1M, 2.5|iM and 5|iM) in DMEM at 37°C for 2 days.2. To investigate the disaggregation effect of gastrodin on preformed AB fibril, 1^M A^42 oligomer was pre-incubated at 37°C for 2 days to form AB fibril, followed by incubation with the same gradients of gastrodin for an additional 3 days at 37°C.3. For both assays, AB42 were incubated with PBS (DMEM) under the same conditions as controls. The samples were then measured by adding 5yM thioflavine T (ThT) solution. Fluorescence intensity was monitored at an excitation wavelength of 450nm and an emission wavelength of482nm by a spectrometer (Synergy H4, Bio Tek). Each experiment was performed in triplicate and the means of the triplicates were used for statistical analysis. The experiment was repeated three times in separate laboratories.Inhibition assay of A6 aggregationby Western blotting. A total of 1^M AB42 oligomer was incubated with different concentrations of gastrodin (O^M, 0.3^M, 1.0^M and 3.0^M) in DMEM at 37°C for 2 days. The resultant solutions were mixed with the same volume of 2x loading buffer without reducing agent, and subjected to electrophoresis(电泳).AB42 oligomer without pre-incubation was set as an additional control.Transmission electron microscopy negative staining.To further validate the effect of gastrodinon AB fibrillization and fibril disaggregation, transmission electron microscopy (TEM) negative staining was performed. The incubation procedures of A^42 and gastrodin were the same as ThT assay.Copper grids were pre-placed on the bottom of wells in a 24- well plate and incubated with AB in the presence or absence of gastrodin. Thereafter, the peptide was stained with 2% aqueous phosphotungstic acid for 30 seconds. Samples were examined using a Joel 1200 EX transmission electron microscopy equipped with Megaview 3 Digital Camera.SH-SY5YCells culture, cell viability assay and neurites outgrowth assay.To investigate the antagonistic effects of gastrodin against AB toxicity in vitro, human neuroblastoma cell line SH-SY5Y was cultured in growth medium in 5% CO2humified incubator at 37 °C. For the cell viability assay, MTT assays was performed as described previously [1]. Briefly, SH-SY5Y cells were treated with 1^M AB with or without Edaravone (0.3^M, 1^M or 3^M) for 24 hour, followed by incubation with MTT (0.5 mg/ml) for 4 h and 10% SDS solution for 15 min at 37 ° C. The optical density at 563 nm was evaluated by a microplate reader (Synergy H4, Bio Tek). For neurites outgrowth, SH-SY5Y cells were cultured for 7 days in a medium with 1% FBS and 10 |iM all-trans-retinoic acid (RA) (Sigma, US), followed by the inbubation with 1 |iM AB with or without GA at different concention mentioned above for 24 hours. The cell images were taken by microscopy, the length of five longest neurites per view field were measured and data from six view fields per group were analysed.Primary cortical neurons culture, ROS assay and propidium iodide (PI) labeling . Primary cortical neurons were isolated from new born 129sv mice brain and cultured on poly-D-lysine precoated coverslips. After 72-hour seeding, the cells were treated with 1^M AB42 oligomers with or without Edaravone at different concentration (0.3, 1or 3^M) for 24 hours and then cells were fixed with 4% paraformaldehyde for 10 min and followed with MAP-2 and DAPI staining. The length of axons was measured using ImagJ software. After different treatments, the cortical neurons were subjected to ROS assay according to the protocol provided by the manufacturer (Cell BiolabsInc, Cat STA-347). The samples were read and quantified with Fluostar OPTIMA from BMG Labtech. Primary cortical neurons after 48 hours culture were also stained in live with propidium iodide (PI). Briefly, live neurons were washed with warm PBS for 5 mins for 3 times and then treated with PI diluted (2 yg/ml) in the HEPES buffer containing100 mM HEPES, 140 mM NaCl, 25 mM CaCl2, pH 7.4 for 15 mins at room te。