Fluostar optima analyzer

According to Bacon and colleagues, the SG molecule intercalates in the genomic DNA of the malaria parasite and then fluorescence [1 (link)]. In order to demonstrate this, the relationship between parasite DNA and SG fluorescence was examined. Briefly, P. falciparum DNA from a 10 % parasite culture was extracted using standard method. After determining the concentration of the parasite DNA it was serially diluted with DNase-free water to generate a concentration range. One hundred microlitres of each concentration was dispensed into a 96-well plate followed by the addition of 100 μl lysis buffer [20 mM Tris (pH 7.5), 5 mM EDTA, 0.008 % (W/V) saponin, and 0.08 % (V/V) Triton X-100] containing SG (1× final concentration)—here in referred to as LBS. Control wells consisting of 100 μl Dnase free water and equal volume of LBS were also set up. Plates were incubated in the dark at room temperature for 1 h and the fluorescence intensity was measured at 485 nm excitation and 528 nm emission using a BMG labtech FluoStar optima analyzer. Fluorescence units were then plotted against parasite DNA concentration. Each data point was performed in triplicate and three independent experiments were performed.

For human HNSCC cell line analysis, cells were seeded at 1x10⁴ cells/well, were left to rest for 24 hours in 37°C and infected with 1, 10, 100 and 1000 VP/cell of Ad5/3-E2F-D24-hTNFa-IRES-hIL-2 or Ad5/3-E2F-D24. Then, the cell viability of the HNSCC cell lines was determined by MTS on day 2, 6, 10. Wells were incubated for 2 hours with 40 µl of CellTiter 96 AQueous One Solution Proliferation Assay reagent (Promega, Wisconsin, USA). Absorbance was read at 492 nm using a Fluostar OPTIMA analyzer (BMG Labtech, Offenburg, Germany). Each time data-point was normalized to the vehicle control group.