Cell viability is determined by a modified MTS assay, which is based on the conversion of Tetrazolium salt by mitochondrial dehydrogenase to a formazan product spectrophotometrically measurable at 490 nm. PC12 cells are plated in 96-well plates at a density of 10 000 cells/well and maintained for 16 h in complete medium. Cells are then incubated in the absence (control) and presence of 40 μM Aβ(31-35) and Aβ(25-35) with reduced, oxidized and norleucine-substituted methionine-35 staurosporine 10 μM is used as positive control of 100% of cellular death. After 48 h of peptide-incubation, 20 μL of MTS reagent (2.0 mg/mL) is added to each well. The cells are then incubated for 30-45 min at 37 °C in a 5% CO₂ incubator. The reaction is stopped by adding 25 μL of 10% SDS. The plates are read with a microplate reader at 490 nm. Each data point is obtained using a triplet-well assay^[2].

MCE has not independently confirmed the accuracy of these methods. They are for reference only.

Rats^[3]
Fifty-four Male Sprague-Dawley rats (2 months old, 300-350 g) are used. Amyloid beta-peptide(25-35) is dissolved in sterile distilled water at a concentration of 1 mg/mL as a stocking solution. Animals are infused with 5 μL/side of sterile distilled water (control), aggregated Aβ_25-35 (2 μg/μL), into bilateral cerebral lateral ventricles at a rate of 1 μL/min; the needle is left in place for 5 min. Then the needles are removed and rats are kept on a warm pad until they are awakened. To determine the neuroprotective effect on AD rats, the Aβ_25-35 treated rats are treated with CDS of different doses and Donepezil once daily for 23 days (including duration of behavior test). Experiment is performed to test the effect of CDS on Aβ_25-35-induced memory impairment using Morris water-maze and step-through passive avoidance tasks. Specifically, all of the rats are randomly divided into 6 groups for the experiment: (a) Vehicle 1 (for Aβ_25-35)+vehicle 2 (for CDS and Donepezil), (b) Aβ_25-35+vehicle 2, (c) Aβ_25-35+CDS (130 mg/kg), (d) Aβ_25-35+CDS (260 mg/kg), (e) Aβ_25-35+CDS (520 mg/kg), (f) Aβ_25-35+Donepezil (0.5 mg/kg). One day after cerebroventricular microinfusions of Aβ_25-35 (10 μg/side) or its vehicle, rats are treated (i.g.) with CDS or Donepezil or vehicle 2, once daily for 14 days prior to the beginning of Morris water maze, followed by passive avoidance task.

MCE has not independently confirmed the accuracy of these methods. They are for reference only.

• [1]. D'Ursi AM, et al. Solution structure of amyloid beta-peptide (25-35) in different media. J Med Chem. 2004 Aug 12;47(17):4231-8.  [Content Brief]

  [2]. Clementi ME, et al. Abeta(31-35) and Abeta(25-35) fragments of amyloid beta-protein induce cellular death throughapoptotic signals: Role of the redox state of methionine-35. FEBS Lett. 2005 May 23;579(13):2913-8.  [Content Brief]

  [3]. Liu M, et al. Cognitive improvement of compound danshen in an Aβ25-35 peptide-induced rat model of Alzheimer's disease. BMC Complement Altern Med. 2015 Oct 23;15:382.  [Content Brief]

  [4]. Patel NS, et al. Potent anti-angiogenic motifs within the Alzheimer beta-amyloid peptide. Amyloid. 2008;15(1):5-19.  [Content Brief]

  [5]. Julia Fedotova, et al. Ropren? is a polyprenol preparation from coniferous plants that ameliorates cognitive deficiency in a rat model of beta-amyloid peptide-(25–35)-induced amnesia. Phytomedicine. 2012 Mar 15;19(5):451-6.  [Content Brief]