Innova 4080 incubator shaker

HFIP-treated Aβ42 peptide films were prepared in phosphate buffer at a nominal concentration of 1 mg/ml on ice and added to 96-well optical-bottom microtiter plates (Thermo Fisher Scientific, Rochester NY), followed by 1.6 µl of ThT at a concentration of 5 mM in phosphate buffer. Phosphate buffer was added to the wells to produce a final volume of 200 µl and final Aβ and ThT concentrations of 20 and 40 µM, respectively. The solutions were mixed gently by pipetting solution into and out of the pipette tip. The plates were sealed using an adhesive plate sealer and incubated at 37°C with gentle shaking (160 rpm) (Innova 4080 incubator shaker, New Brunswick Scientific, NJ). Fluorescence was determined using λ_ex=450 nm and λ_em=482 nm. Readings were taken immediately (0 h), every hour for 6 h, and subsequently at 24 h intervals. Buffer alone, instead of peptide, constituted the ThT blank. A minimum of 5 replicates of each sample was measured. The mean of the blank readings were subtracted from the mean of the sample readings at each time point and the corrected values, along with mean and SD, were plotted using KaleidaGraph (v 4.1, Synergy Software, Reading, PA). Statistical analyses (t-test and Mann-Whitney Rank test) were performed using SigmaStat (Jandel Scientific, San Jose, CA).

Aβ42, iAβ42 and Ac-iAβ42 peptide solutions were prepared as stated in “Thioflavin T (ThT) binding.” The peptides then were incubated at 37°C with gentle shaking in an Innova 4080 incubator shaker (New Brunswick Scientific, Edison, NJ). CD spectra were obtained every 30 min for the first 2 h, and subsequently every hour, using a JASCO J-810 spectropolarimeter (Tokyo, Japan). The CD parameters were: wavelength scan range, 190–260 nm; data pitch, 0.2 nm; continuous scan mode, 10 scans of each sample; scan speed, 100 nm/min; 1 sec response; and band width, 2 nm. The spectra were processed using the means movement smoothing parameter within the Spectra Manager software. The data were subsequently plotted using KaleidaGraph (v 4.1.3).