I would to study apoptosis, but I haven't regular access flow cytometry to use Annexin V/PI staining. Could suggest me sensitive methods to investigate apoptosis?
Hello Priscilla Chiofalo
There are many methods besides flow cytometry to investigate apoptosis. I have mentioned a few of them below.
Most apoptotic process involves the activation of a cascade of caspases that mediate upstream signaling events ultimately leading to the activation of executioner caspases involved with destroying the cell. Executioner caspases are involved with the terminal proteolytic events of the apoptotic process. Those events include breaking down the cytoskeleton that leads to morphological changes observed during apoptosis and inactivating repair enzymes such as poly ADP ribose polymerase (PARP) to be cleaved by caspase. You could try Western Blot analysis of your sample for pro- and active caspase as well as cleaved PARP.
Measuring caspase-3/7 activity is one of the assay to detect the process of apoptosis which you could try. You may measure caspase-3/7 activity using consensus tetrapeptide substrates in lytic cell-based assay to generate a fluorescent or luminescent signal measured with a plate reader.
You could also try DNA fragmentation assays. You know that apoptosis and DNA cleavage go hand in hand. So you could determine the extent of apoptosis by measuring the DNA fragments present in your sample. You could make use of apoptotic DNA ladder analysis, ELISA quantification and TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling) assays for this purpose.
There are two common assays for early apoptotic events namely, the mitochondrial membrane potential assay and the cytochrome c assay. Early in apoptosis, pores open in mitochondrial membranes, making them permeable to ions and other molecules. The resulting depolarization of the mitochondrial membranes can be assayed using fluorescent indicator dyes. In addition, when pore formation disrupts mitochondrial membrane potential, cytochrome c is released from mitochondria into the cytoplasm. The cytochrome c assay quantifies cytochrome c in the cytosolic versus mitochondrial extracts.
Apoptosis shows membrane blebbing. So you could use light microscopy analysis to study membrane blebbing.
In this way, using the above methods (to mention a few of them), you can study apoptosis without flow cytometry.
Best.
Hello @Priscilla Chiofalo,
If you have accessibility of fluorescence microscopy then you can do AO-EtBr staining assay to determine apoptosis. If you want to know more about this assay then dm me .. i will share protocol or you can read below articles.
1. https://doi.org/10.1016/j.colsurfa.2020.125501
2. https://doi.org/10.1016/j.ijbiomac.2022.02.031
3. https://doi.org/10.1021/acsomega.2c01041
You can use annexin V/PI kit to observe apoptosis by fluorescence microscope.
Hi Priscilla Chiofalo
You can find several commercially available kits that would help you in imaging apoptotic cells. Imaging is the most widely used method other than flow cytometry. Please find the links to protocols and kits attached.
1. https://www.thermofisher.com/in/en/home/life-science/cell-analysis/cellular-imaging/fluorescence-microscopy-and-immunofluorescence-if/imaging-apoptosis-assays.html
2. https://www.takarabio.com/products/gene-function/cell-biology-assays/apoptosis-detection-kits/in-situ-apoptosis-detection
3. https://www.abcam.com/products/assay-kits/apoptosis-necrosis-assaykit-blue-red-green-ab176750.html
Thanks,
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