Category: Assay Development and Screening
Apoptosis is an indispensable process for normal tissue development and homeostasis, which allows cells to undergo timely programmed cell death. When apoptosis is induced, in most cases, caspase-3/7 is activated and the cell membrane is compromised by rearrangement of phospholipid. As activation of caspase-3/7 always leads to apoptotic death of the cells, the caspase activity is considered to be a reliable apoptosis marker. In addition, externalization of phosphatidylserine (PS) in the cell membrane is also a typical apoptosis marker. Therefore, the two markers have been assessed by enzymatic and flow cytometric endpoint assays. However, major disadvantages of these assays are (1) it obtains only a single result for each set of endpoints, and therefore (2) it is impossible for real-time measurement of live cells. In order to overcome these disadvantages, we combined bioluminescence and fluorescence microscopy and tried to detect the apoptotic to necrotic process on the same live cell samples.
Firstly, we created a stable U2OS cell line that expresses Luc2 luciferase (Promega corp.). To detect caspase-3/7 activity, the aminoluciferin incorporating the DEVD (Asp-Glu-Val-Asp) motif recognized by the caspase-3/7 (VivoGlo™ Caspase-3/7 substrate, Promega corp.) was added into the culture medium. When caspase-3/7 is activated, liberated luciferin reacts with Luc2 and generates bioluminescent light (lmax=609 nm).
Secondary, to detect externalization of PS and secondary necrosis (late apoptosis), we applied RealTime-Glo™ Annexin V Apoptosis and Necrosis Assay Kit (Promega corp.) to the U2OS stable cell line. This kit contains equal ratios of two annexin V molecules expressed as fusions with large or small subunits of NanoBiT luciferase (Promega corp.) and time-released substrate. When PS is externalized on the cell membrane, annexin V binds to PS as a dim on the cell surface and NanoBiT Luciferase is reconstructed, and bioluminescence light (lmax=463 nm) is generated. This kit also contains a cell-impermeant, profluorescent DNA dye, which detects necrosis by fluorescent light (lmax=525nm) with the dye binding to nuclear DNA.
Finally, we observed apoptosis and necrosis of the U2OS cells by sequential bioluminescence and fluorescence imaging using a microscope, LV200 (OLYMPUS) with induction of apoptosis by staurosporine (STS). Apoptotic processes were observed as caspase-3/7 activation in single cells and externalization of PS on cell surface by bioluminescence images, and secondary necrosis as fluorescence image of the nucleus together with morphological changes of cell components.
RYUTARO AKIYOSHI– Senior Supervisor, OLYMPUS Corporation, Hachioji-shi, Tokyo, Japan