how to detect and counted early apoptotic, late apoptotic and necrotic cell population using confocal microscopy?
Is flow cytometry is mandatory to count the population or without using flow cytometry the cell polulations can be counted using confocal microscopy?
For image analysis, confocal microscopy is best of course.
In apoptotic cells, the membrane phospholipid phosphatidylserine (PS) is translocated from the inner to the outer leaflet of the plasma membrane, thereby exposing PS to the external cellular environment. Annexin V is a Ca2+ dependent phospholipid-binding protein that has a high affinity for PS. Since annexin V staining precedes the loss of membrane integrity which accompanies the later stages of cell death resulting from either apoptotic or necrotic processes, staining with annexin V-FITC is typically used in conjunction with a dye such as PI allows to identify early apoptotic cells (PI negative, annexin V-FITC positive) from dead cells (PI positive, annexin V-FITC positive). you can see this picture for easy understanding- https://www.creative-bioarray.com/support/annexin-v-apoptosis-assay.htm
If you want to distinguish late apoptotic cells from necrotic ones you have to use flow cytometry. You can also check this for reference
Article 9-phenyl acridine photosensitizes A375 cells to UVA radiation
Hi!
Yes, you can use immunofluorescence microscopy to evaluate the stages of cell death. Is good to keep in mind that dying cells present different morphological and biochemical features and these features are different from apoptotic and necrotic cells. So for confocal microscopy, I would recommend using nuclear staining to detect nucleus morphology and chromatin compaction/relaxation and fragmentation combined with some caspase staining.
In necrotic processes, there is no caspase activation, and nuclear configuration can give you an indication of apoptosis or necrosis process.
You can make a time point kinetics to have a comprehensive idea about which mechanism is taken place in your studies. Use good controls and it would be fine.
A comment about Annexin V is that not always PS exposure is related to early apoptosis. PS exposure is common after cell activation/ stimulation.
I also recommend this paper here Article Measurement of Cell Death in Mammalian Cells
All the best!
- Badges
- Science topic
- Similar topics
- Philosophy
- Philosophy Of Science
- Reasoning