I have done by counting the distorted nuclei manually but that does not seems to be accurate as we fail to count it properly
Hi,
I lack a little information to be sure what you did exactly but I try to answer as thoroughly as possible.
DAPI should not permeate the cell membrane of a healthy (non fixed) cell, thus it can be used as a marker for necrotic/apoptotic cells. But you can not differentiate between necrosis or apoptosis, for this you normally use something like Annexin V that stains PS which is externalized upon the beginning of apoptosis.
So you take your whole population and take only DAPI positive cells as necrotic, DAPI plus Annexin V as apoptotic and only Annexin V positiv cells as early apoptotic.
Hope this helps you,
Jürgen
Hello,
Dapi will enter the membrane also of intact cells (non fixed) as well as Hoechst! It depends on concentration and incubation time. You have to use about 10 µg/mL (5 min) for live cell count than it will enter also the intact membrane....With a lower concentration and a shorter incubation time you will possible stain your apoptitic cells...
Best regards,
Constanze
DAPI (4',6-diamidino-2-phenylindole) is a DNA-specific probe which forms a fluorescent complex by attaching in the minor grove of A-T rich sequences of DNA. It also forms nonfluorescent intercalative complexes with double-stranded nucleic acids.
DAPI visualizes nuclear DNA in both living and fixed cells. DAPI staining has been used to determine the number of nuclei and to assess gross cell morphology. It is rough marker for apoptosis. See the link:
DAPI as a useful stain for nuclear quantitation. . Biotech Histochem 1991;66(6):297-302.
Article DAPI as a Useful Stain for Nuclear Quantitation
Indeed, its not very easy to differentiate apoptotic cell death from non-apoptotic cell death just on the basis of counting DAPI positive or negative nuclei straight forward. Hence, more sensitive assay like (TUNEL assay) may be used to support the morphological changes. DAPI staning may just guide you if the cells showing smaller and more brightly stained nuclei could guide you that the cell is dying. Indeed, DAPI staining intensify the florescence in a cells that show apoptotic chromatin condensation. Hence, the Hoechst could be always better in place of DAPI.
Thus, in order to confirm the data generated from DAPI experiment, you may have to support your data with other authentic markers like caspases activity through ICC and WB in the cell lysates and finally by DNA ladder.
Best
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