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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.

During summer 2012 Shell performed exploratory drilling at Sivulliq, a lease holding located in the autumn migration corridor of bowhead whales (Balaena mysticetus), northwest of Camden Bay in the Beaufort Sea. The drilling operation involved a number of vessels performing various activities, such as towing the drill rig, anchor handling, and drilling. Acoustic data were collected with six arrays of directional recorders (DASARs) deployed on the seafloor over ~7 weeks in Aug-Oct. Whale calls produced within 2 km of each DASAR were identified and localized using triangulation. A "tone index" was defined to quantify the presence and amplitude of tonal sounds from industrial machinery. The presence of airgun pulses originating from distant seismic operations was also quantified. For each 10-min period at each of the 40 recorders, the number of whale calls localized was matched with the "dose" of industrial sound received, and the relationship between calling rates and industrial sound was modeled using negative binomial regression. The analysis showed that with increasing tone levels, bowhead whale calling rates initially increased, peaked, and then decreased. This dual behavioral response is similar to that described for bowhead whales and airgun pulses in earlier work. Increasing call repetition rates can be a viable strategy for combating decreased detectability of signals arising from moderate increases in background noise. Meanwhile, as noise increases, the benefits of calling may decrease because information transfer becomes increasingly error-prone, and at some point calling may no longer be worth the effort.

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.

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.

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.

3-hydroxyphenylacetylene (3-HPA) served as a novel, activity-dependent, fluorogenic and chromogenic probe for bacterial enzymes known to degrade toluene via meta ring fission of the intermediate, 3-methylcatechol. By this direct physiological analysis, cells grown with an aromatic substrate to induce the synthesis of toluene-degrading enzymes were fluorescently labeled.

A thick sequence of floodplain sediments has accumulated in the Delaware River Valley by the process of overbank deposition. Textures in the sediments indicate that the sequence contains no point-bar deposits and is unbroken by periods of erosion. Fourteen radiocarbon dates show that deposition began at least 6000 years ago and has continued to the present. Because the Delaware River shifts its position laterally at a very slow rate, overbank deposition becomes dominant in the construction of its floodplain.