Where as in case of MTT assay, the results were positive, but in DNA fragmentation assay, their is no fragmentation of genomic DNA can be seen. So, my question is, is the DNA fragmentation solely require to prove apoptosis has occured? Or their may be several other alternative factors to check ?
Kindly, mention some assays which supports as a evidence of apoptosis.
Thank you.
MTT assay does not discriminate apoptosis from cell cycle arrest. TUNEL assay and cytochrome-C release from mitochondria are needed to check apoptosis.
Jannatul firdous Siddique AnnexinV assay for early apoptosis.
You can check it through Tunnel method and AnnexinV-FITC assay & hoechst staining method is also a way to determine apoptosis.
DNA fragmentation occurs in late stage of apoptosis. Even some drug induce apoptosis but we will not be able to detect DNA fragmentation.
Early or late apoptosis can be detected by Annexin V/PI staining while DNA fragmentation can be visualize by Tunnel assay/Hoechst staining either by fluorescence microscopy or by flow cytometry.
For HOECHST staining you can follow the below article:
Article Chetomin induces apoptosis in human triple-negative breast c...
You can check and confirm by cell cycle analysIs. As DNA content changes. It will help for both cell cycle and apoptosiS.
thank you so much for all the answers above. what if i go for AO/EB assay, how it will help?
Jannatul firdous Siddique you can use AO/EB, however Annexin V/PI and Hoechst or TUNEL give more accurate information about cell death.
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