Enzyme linked immune sorbent assay kits

The determinations of p53, Bax, Bcl-2, and cytochrome C proteins were done using enzyme-linked immune-sorbent assay kits (R&D Systems, Minneapolis, MN, USA) according to the manufacturer’s instructions. The protein levels were determined in 1 × 10⁴ MDA-MB-231 cells treated with 5, 10 and 50 μg/mL mango kernel extract for 12 and 24 h, and the absorbance values were read on a microplate reader. The results were presented as fold change in protein expression relative to the untreated cells.

Caco-2 cell monolayers grown in 12-well plates (Corning Incorporated, Kennebunk, ME, USA) were serum-starved overnight, incubated in the presence of (0,1, 10, 100 µM) trans-ferulic acid or 1/2 volume of culture supernatants from Bacteroides intestinalis, and after 1 h, Escherichia coli lipopolysaccharide (LPS) was added at a final concentration of 10 µg/mL. Culture media were collected 24 h after ferulic acid administration to determine IL-12p70, interferon (IFN)α, and IFNβ by commercially available enzyme-linked immune sorbent assay kits (R&D Systems, Minneapolis, MN). Total RNA from Caco-2 cells at 24 h was isolated using Sepasol-RNA I Super G reagent (Nacalai). The RNA concentration and purity were determined by UV absorption at 260:280 nm using an Ultrospec 1100 pro UV/Vis spectrophotometer (Amersham Biosciences, NJ). RNA was reverse-transcribed into cDNA using a ReverTra Ace qPCR RT kit (TOYOBO, Osaka, Japan), according to the manufacturer’s protocol, and then the DNA was amplified by PCR using Quick Taq HS DyeMix (TOYOBO). The sequences of the primers and the number of PCR cycles are listed in Table 1. The PCR products were separated on a 2% agarose gel containing 0.01% ethidium bromide. The amount of mRNA was normalized against the glyceraldehyde 3-phosphate dehydrogenase mRNA.