Bioluminescence involves light emission by live organisms and has been preserved throughout evolution mainly in marine organisms and some insects, bacteria, and fungi. It is caused by the oxidation of a small organic molecule, luciferin, with molecular oxygen, which is catalysed by the enzyme luciferase. Reaction results in the conversion of luciferin to oxyluciferin in an excited state. Oxyluciferin emits visible light and then returns to the ground state. Luciferase is an adenosine triphosphate dependent enzyme and is activated by metal ions (typically Mg2+). Bioluminescence imaging (BLI) is mainly based on the firefly luciferase – luciferin system and has been widely used for analytical monitoring and exploration of a plethora of biological processes in vitro and in vivo. The objective of this work was to evaluate the relevance of physicochemical parameters for light emission. We have defined concentrations of cofactors and other physicochemical properties for optimizing application of bioluminescent reaction in vitro. We have verified our findings in cell-based assays carried out on luciferase-expressing 4T1 triple-negative breast cancer cell line derived from the mammary gland tissue of a mouse BALB/c strain. Finally, we have studied bioluminescent reaction in a mouse model of breast cancer after the orthotopic implantation of mammary gland adenocarcinoma 4T1 cells genetically labelled with luciferase. We have compared bioluminescent imaging to traditional methods of tumor measurements using calipers. The use of bioluminescence imaging offers great advantages over using caliper measurement and enables visualization of tumor initiation, progression, metastasis, and tumor microenvironment in longitudinal studies for estimation of mammary tumors. The purpose of this work was to optimize application of bioluminescent imaging for in vitro and in vivo biomedical research and development of new drugs, therapies, and diagnostic methods.
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