Right atria or sinus nodal cells are homogenized in lysis buffer consisting of (50 mM Tris-HCl pH 7.5, 100 mM KCl, 1 mM ethylenediamine tetraacetic acid, 1 mM ethylene glycol tetraacetic acid, 1 mM dithiothreitol, 0.1 mM phenylmethylsulfonyl fluoride, 0.5 mM Benzamidine, 20 mg/L Leupeptin, 20 mM sodium pyrophosphate, 50 mM NaF, and 50 mM sodium β-glycerophosphate), and total protein content is determined by the Bradford assay. Caspase-3 activity is determined by EnzChek Caspase-3 Assay Kit^[3].

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

Ovarian cancer cells are seeded in 96-well plates at 2000 cells/well and incubated overnight before treatment with 0-160 μM Kaempferol for 24 hours in triplicates. The medium is removed, and the plates are freeze-thawed to lyse cells. Each well is added with 200 μL 1× CyQUANT cell lysis buffer containing 5x SYBR Green I and incubated at room temperature (RT) for 5 minutes. The reaction (50 μL) is transferred to PCR strip tubes and the fluorescent signal is measured at 90°C with a real-time Chromo4 PCR instrument. To ensure that cell proliferation assays are performed within a linear range of cell numbers, a standard curve is generated by seeding different amount of OVCAR-3 cells (based on counting with a hemacytometer) in a 96-well plate, and measuring genomic DNA abundance after overnight incubation. Three independent experiments are performed and data is pooled for statistical analysis^[2].

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

Mice^[4]
Three-week-old male BALB/c mice are randomly assigned to the four treatment groups as follows (n=8 per group). (1) PBS-sensitized mice; (2) BSA-sensitized mice; (3) BSA-sensitized and 10 mg/kg Kaempferol-administered mice; and (4) BSA-sensitized and 20 mg/kg Kaempferol-administered mice. Mice are given a commercial mouse chow diet containing 20.5% protein, 3.5% fat, 8% fiber, 8% ash, and 0.5% phosphorus and are allowed access to food and water ad libitum. The mice are kept under a 12 h light and dark cycle at 23±1°C with 50%±5% relative humidity in specific pathogen-free conditions. Mice are allowed to become accustomed to their surroundings for one week before starting the allergic experiments. Sensitization of all experimental mice is carried out by subcutaneous injection with 20 μg BSA in 30 μL PBS and 50 μL Imject Alum on days 0 and 14. The control mice are injected with a combination of 50 μL PBS and 50 μL Imject Alum without BSA. On days 28, 29, and 30, only the experimental mice sensitized to BSA are subject to inhalation of 5% BSA, while control mice are challenged with 5% PBS for 20 min in a plastic chamber connected to a Medel aerosol nebulizer. All mice are sacrificed 24 h after the last challenge. Whole blood samples are directly used to measure the contents of eosinophils, basophils and neutrophils. The right lung is stored in 4% paraformaldehyde until use.

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

• [1]. Luo H, et al. Kaempferol nanoparticles achieve strong and selective inhibition of ovarian cancer cell viability. Int J Nanomedicine. 2012; 7: 3951-3959.  [Content Brief]

  [2]. Luo H, et al. Kaempferol induces apoptosis in ovarian cancer cells through activating p53 in the intrinsic pathway. Food Chem. 2011 September 15; 128(2): 513-519.  [Content Brief]

  [3]. An M, et al. Protective effects of Kaempferol against cardiac sinus node dysfunction via CaMKII deoxidization. Anat Cell Biol. 2015 Dec;48(4):235-43.  [Content Brief]

  [4]. Shin D, et al. Dietary Compound Kaempferol Inhibits Airway Thickening Induced by Allergic Reaction in a Bovine Serum Albumin-Induced Model of Asthma. Int J Mol Sci. 2015 Dec 16;16(12):29980-95.  [Content Brief]