MitoSOX-based assays are widely used to detect mitochondrial reactive oxygen species (ROS), especially superoxide. To this end, 5 μM MitoSOX is commonly used. In this ROS Protocols article, we described the flow cytometric protocol involving the use of various concentrations of MitoSOX (1, 2.5, 5 μM) for detecting mitochondrial ROS in control and mitochondrial DNA-deficient (MD) melanoma B16-F10 cells. We also compared the MitoSOX-based flow cytometry with lucigenin-derived chemiluminometry for their ability to reliably detect the relative differences in mitochondrial ROS formation in the control and MD cells. Our results suggested that 1 μM, rather than the commonly used 5 μM, appeared to be the optimal concentration of MitoSOX for detecting mitochondrial ROS via flow cytometry.

Detection and measurement of doxorubicin in biological systems, including body fluids, cells, and tissues, are instrumental in understanding the mechanisms of action of this widely used drug in treating cancer as well as in causing adverse effects. In this article, we, for the first time, characterized the use of fluorescence-based techniques, including fluorescence spectrometry, microscopy, and flow cytometry in measuring and/or detecting doxorubicin in biological systems, including cell lysates and cultured intact cells. We showed that doxorubicin has a maximum excitation and emission wavelength of 470 and 560 nm, respectively. The detection sensitivity by fluorescence spectrometry is less than 0.1 μM in buffers and cell lysates. Fluorescence microscopy demonstrated the readily detection of concentration-dependent accumulation of doxorubicin in cultured cells via either green or red fluorescence, but with green fluorescence showing a higher sensitivity of detection. Flow cytometry also revealed sensitive detection of doxorubicin accumulation in cell suspensions in a concentration-dependent manner. The readily and sensitive measurement and detection of doxorubicin by the above three fluorescence-based techniques has important implications in studying the cellular dynamics of doxorubicin in both cancer and normal cells under various experimental conditions.

In vivo imaging of cancer cell growth and invasion is instrumental in studying cancer cell behavior and in developing effective anticancer agents. In this ROS Protocols article, we report the experimental protocol and steps involving the implantation of luciferase-expressing Lewis lung carcinoma (LLC) cells in normal syngeneic C57BL/6 mice. Using the Berthold NightOwl LB981 in vivo imaging system, we observe the time-dependent growth and invasion of the lung cancer cells following subcutaneous injection of luciferase-expressing LLC cells. The three-dimensional image and counts of photon emission of the tumor mass are obtained to estimate the relative size of the tumor. Ex vivo imaging of the isolated lungs supplemented with D-luciferin and adenosine triphosphate (ATP) is obtained to determine lung metastasis of the LLC cells. The LLC cell load in entire mouse lungs is further determined by quantitative bioluminometry with a concurrently run standard curve of the number of LLC cells versus bioluminescence intensity. This in vivo imaging system in live mice, in combination with ex vivo imaging of isolated lungs as well as quantitative bioluminometry of target tissues, may provide important information on the in vivo cancer cell dynamics in immunocompetent syngeneic C57BL/6 mice and offer a valuable tool for studying experimental anticancer agents, including redox-modulating compounds, which are promising anticancer modalities.

Paraoxonase (PON) refers to a family of three enzymes, namely PON1, PON2, and PON3. PON1 and PON3 are found in circulation bound to high-density lipoprotein, whereas PON2 is an intracellular protein. PON1 was first discovered as an enzyme to hydrolyze the organophosphate pesticide paraoxon, an activity that both PON2 and PON3 lack. All three PON enzymes are able to degrade oxidized lipids and protect against oxidative stress. PON enzymes also act to suppress inflammation. Animal studies show a critical role for PON enzymes, especially PON1 in protecting against cardiovascular diseases and related disorders, including diabetes and metabolic syndrome. In line with the findings in experimental animals, accumulating evidence from clinical research also indicates that PON enzymes function as potential protectors in human cardiovascular diseases and related disorders. Identification of PON enzymes as important players in cardiovascular health will facilitate the development of novel preventive and therapeutic modalities targeting PON enzymes to combat cardiovascular diseases and related disorders, which collectively constitute the chief contributors to the global burden of disease. This review describes the biochemical properties and molecular regulation of PON and summarizes the major recent findings on the functions of PON in protecting against cardiovascular diseases and related disorders.

Animal models are essential for developing effective drugs for treating human cancer. Examination of the formation of lung surface foci of B16-F10 melanoma cells is a widely used animal model for studying cancer metastasis and drug intervention. This model, however, suffers from several drawbacks, including its non-quantitative nature and inability to yield information on cancer cell load inside the target organ. Here we report the development of a highly sensitive, bioluminescence-based method for quantifying melanoma cell load in mouse lungs following intravenous injection of luciferase-expressing B16-F10 melanoma cells. This method could readily detect as few as 1–10 cells in the samples and enable quantification of cancer cell load before the formation of surface foci in mouse lungs following metastasis of intravenously inoculated B16-F10 melanoma cells. This innovative bioluminometry-based method has important implications for studying anticancer drugs, including naturally occurring redox-active quinones that generate reactive oxygen species to kill cancer cells.