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1、C On-line Methods Preparation of oligomer form of A42.Synthetic A42 were purchased from American Peptide (Sunnyvale, CA, USA) and prepared following the protocols described previously. 1The A42peptide was dissolved in 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP, Sigma) at 1 mg/ml concentration, and was
2、aliquoted in Eppendorf tubes (100 g/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 A42( MW:4514.08), the dried pallet was re-suspended in DMEM
3、or PBS to a final concentration of 100 M , followed by incubation at 4C for 24 hours. The oligomer form of A42 was checked by Western blot before use. (add 221.5 L DMEM) The thioflavin T (ThT) fluorescence assay. 1. To test the inhibitory effect of gastrodin on A aggregation, 1 M A42 (4. 514 g ) was
4、 incubated with a serial gradient of gastrodin (0 M, 0.156 M, 0.313 M, 0.625 M, 1.125 M, 2.5 M and 5 M) in DMEM at 37C for 2 days. 2.To investigate the disaggregation effect of gastrodin on preformed A fibril, 1 M A 42 oligomer was pre- incubated at 37 C for 2 days to form A fibril, followed by incu
5、bation with the same gradients of gastrodin for an additional 3 days at 37C. 3.For both assays, A 42 were incubated with PBS（ DMEM） under the same conditions as controls. The samples were then measured by adding 5 M thioflavine T (ThT) solution. Fluorescence intensity was monitored at an excitation
6、wavelength of 450nm and an emission wavelength of 482nm 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 A ag
7、gregationby Western blotting. A total of 1 M A 42 oligomer was incubated with different concentrations of gastrodin (0 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 electrop
8、horesis( 电泳 ). A 42 oligomer without pre-incubation was set as an additional control. Transmission electron microscopy negative staining. To further validate the effect of gastrodin on A fibrillization and fibril disaggregation, transmission electron microscopy (TEM) negative staining was performed.
9、 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 A in the presence or absence of gastrodin. Thereafter, the peptide was stained with 2% aqueous phosphotungstic acid for 30 seconds. S
10、amples 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 A toxicity in vitro , human neuroblastoma cell lin
11、e SH-SY5Y was cultured in growth medium in 5% CO2 humified 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 A with or without Edaravone (0.3 M,1 M or 3 M) for 24 hour, followed by incubation with MTT (0.
12、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 M all-trans-retinoic acid (RA) (Sigma, US), followed by th
13、e inbubation with 1 M A with or withoutGA 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 assa
14、y 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 A 42 oligomers with or without Edaravone at different concentration (0.3, 1or 3 M) f
15、or 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 manufactu
16、rer (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 g/ml) in the HEPES buffer containing 100 mM HEPES, 140 mM NaCl, 25 mM CaCl2, pH 7.4 for 15 mi
