it's cleaved by activated effector Celecoxib caspases

Our confocal imaging tests confirm that subsequent DNV addition, the NucView488 signal is limited to the nucleus or the perinuclear area of cells undergoing apoptosis. Together with data reported by others15, these suggest that the DNV substrate is non fluorescent until it's cleaved by activated effector Celecoxib caspases, thereby allowing the substrate to stain the nuclei of apoptotic cells. Since the DEVD peptide corresponds to the suitable substrate collection for both Caspase 7 and Caspase 3, it is therefore probably cleaved by both enzymes22. This collection could also potentially be cleaved in a slower rate by other members of the Group II family of caspases with somewhat different specificities22. The analysis needs a unique inclusion of DNV substrate, in lack of any washing step.

Additionally, we show the DNV substrate isn't harmful to HeLa cells. Altogether, these observations confirm that a method based on the utilization of the DNV substrate can allow continuous monitoring of caspase activation. Endosymbiotic theory After refining the substrate concentration with HeLa cells, we wanted to confirm the usage of the DNV substrate for live track of apoptosis in high content displays. We demonstrated the NucView488 signal observed in the natural channel could be imaged in high density format over a platform built with an automatic epifluorescence microscope. Imaging of exactly the same well can be performed as many times as needed over the length of a screen, and the images can easily be processed by automatic analysis software and quantified.

Information is gathered at the single cell level, allowing to study heterogeneous populations of cells. We show a high signal is observed and quantified when HeLa Empty cells are treated with Doxorubicin Fostamatinib or Etoposide, both drugs known to induce apoptosis in cancer cell lines. But, pre treatment with a pan caspase chemical may antagonize this large signal, showing the uniqueness of the signal imaged applying the DNV substrate. Of note, we realized that handle cells treated with DMSO exhibit a powerful nuclear staining applying Hoechst 33342, as the nuclear staining for cells pre treated with Doxorubicin and stained with Hoechst in the same conditions is very poor. It's likely that people are observing the quenching of Hoechst fluorescence by energy transfer to Doxorubicin, since the maximum emission wavelength of Hoechst bound to DNA is 458 nm, which will be near the maximum excitation wavelength of Doxorubicin bound to DNA 23.

This probably results in energy transfer between the 2 dyes, which in the quenching of Hoechst fluorescence as formerly observed24, 25. Nuclei staining with the alternative color such as for instance DRAQ5 is therefore recommended when performing nuclei count after doxorubicin treatment. Moreover, in HeLa Bcl XL overexpressing the anti-apoptotic protein Bcl XL, a much lower NucView488 signal was observed when those cells were treated with Doxorubicin.