Mini bis image analysis system

SW-480 cells were plated in 6-well plates at approximately 1 × 10⁶ cells/well and media were replaced with 10% FBS/RPMI. After 24 h incubation, the cells were treated with different concentrations of LER for 24 h. The harvested cells were washed with PBS twice and then lysed with cell lysis buffer (150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% sodium dodecyl sulfate (SDS), 50 mM Tris-HCl, pH 7.5, 2 mM storage) for 30 min at 4°C. The homogenates were centrifuged at 13,000 × g for 10 min to isolate the supernatants. Protein concentrations were determined using the pierce based on bicinchoninic acid protein assay. Thirty grams of the protein from the supernatants were then separated on 10% SDS-polyacrylamide gel and transferred to polyvinylidene difluoride (Bio-Rad) membranes. After transfer, the membrane was blocked at room temperature for 1 h with 5% BSA in Tris-buffered saline-Tween (TBST; 20 mM Tris, 500 mM NaCl, pH 7.5, 0.1% Tween 20). Then, the membranes were incubated with primary antibodies for 1 h, after three washes in TBST for 5 min, followed by incubation with secondary antibody for 1 h at room temperature. The immunoreactive protein bands were detected by using an enhanced chemiluminescence detection system. Blots were developed with a Mini BIS Image Analysis System (DNR Bio-Imaging Systems Ltd., Jerusalem, Israel).

SW-480 cells (1 × 10⁶) were seeded in a 100 mm cell culture dish and incubated in RPMI 1640 for 24 h. After incubation, 200 μg/ml LER was added to the dish and kept for another 24 h. Then, cells were collected and washed with 1 × PBS, resuspended in 100 μl lysis buffer. Ten microliters of RNase (10 mg/ml) was added to the lysate and further incubated for 30 min at 37°C. The DNA samples were electrophoresed in a 1.5% agarose gel in Tris-acetate-EDTA buffer. The DNA bands were visualized by ethidium bromide staining and a Mini BIS image analysis system (DNR Bio-Imaging Systems Ltd., Kiryat Anavim, Israel).

RT-PCR was used to analyze the gene expression in the RAW 264.7 cells, following stimulation with LPS in the presence of different concentrations of SDE for 6 h. The total RNA was isolated with a Trizol reagent in accordance with the manufacturer's instructions (Invitrogen, Carlsbad, CA). The first-strand cDNA was synthesized from the total RNA (2 µg), containing oligo (dT) primers and Moloney murine leukemia virus reverse transcriptase (M-MLV RT, Invitrogen). The primer sequences for iNOS, COX-2, TNF-α, and GAPDH are listed in Table 1. The aliquots of the individual PCR products were separated on 1% agarose gel, stained with ethidium bromide, and imaged using a Mini BIS image analysis system (DNR Bio-Imaging Systems Ltd., Jerusalem, Israel). Densitometric analysis was done using an image analysis software (Quantity One; Bio-Rad, Hercules, CA, USA).