SYBR Green I-induced fluorescence in cultured immune cells: a comparison with Acridine Orange

Using fluorescence microscopy, we explored the ability of cultured immune cells to take up aqueous SYBR Green I (SGI). SYBR Green I, a highly sensitive fluorescent nucleic acid stain, which preferentially binds to dsDNA over ssDNA or RNA with little background fluorescence from unbound molecules. A time course study over 2h using final dilutions of SGI of 1:10,000 and 1:100,000 at 22 and 37 degrees C, revealed the dye quickly entered the cells, stained mitochondrial DNA then nuclear DNA, and SGI-induced green fluorescence increased over time. As staining progressed, heterochromatin appeared as more intense green fluorescent lines, patches and circles against the lower fluorescence of the nucleoplasm. The lower fluorescence from the nucleoplasm indicated SGI also bound to areas of euchromatin. Similar progressive uptake experiments were carried out with the permeant DNA dye Acridine Orange (AO) to provide insight into staining patterns and mode of uptake. Statistical analysis of cells prestained with SGI then tested with Trypan Blue for changes in membrane permeability, revealed no significant difference between controls and treatment for each temperature. It appears that SGI does not compromise cells for up to 2 h following initial exposure.