de Noiron, J.; Hoareau, M.; Colin, J.; Guénal, I. Apoptosis Quantification in Tissue: Development of a Semi-Automatic Protocol and Assessment of Critical Steps of Image Processing. Biomolecules2021, 11, 1523.
de Noiron, J.; Hoareau, M.; Colin, J.; Guénal, I. Apoptosis Quantification in Tissue: Development of a Semi-Automatic Protocol and Assessment of Critical Steps of Image Processing. Biomolecules 2021, 11, 1523.
de Noiron, J.; Hoareau, M.; Colin, J.; Guénal, I. Apoptosis Quantification in Tissue: Development of a Semi-Automatic Protocol and Assessment of Critical Steps of Image Processing. Biomolecules2021, 11, 1523.
de Noiron, J.; Hoareau, M.; Colin, J.; Guénal, I. Apoptosis Quantification in Tissue: Development of a Semi-Automatic Protocol and Assessment of Critical Steps of Image Processing. Biomolecules 2021, 11, 1523.
Abstract
Apoptosis is associated with numerous phenotypical characteristics, and is thus studied with many tools. In this study, we compared two broadly used apoptotic assays: TUNEL and staining with an antibody targeting the activated form of an effector caspase. To compare them, we developed a protocol based on commonly used tools such as filters, zprojection and thresholding. Even though it is commonly used in imageprocessing protocols, thresholding remains a recurring problem. Here we analyzed the impact of processing parameters and readout choice on the accuracy of apoptotic signal quantification. Our results show that TUNEL is quite robust, even if image processing parameters can allow or not to detect subtle differences of the apoptotic rate. On the contrary, images from anticleaved caspase staining are more sensitive to handle and proved to necessitate to be processed more carefully. We then developed an open source Fiji macro automatizing most steps of the image processing and quantification protocol. It is noteworthy that the field of application of this macro is wider than apoptosis as it can perfectly be used to treat and quantify other kind of images.
Keywords
Apoptosis; TUNEL; Caspase; image processing; thresholding; signal quantification; Drosophila
Subject
Biology and Life Sciences, Biochemistry and Molecular Biology
Copyright:
This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
