I would like to stain necrotic cells with ethidium bromide then counter stain with Dapi for estimating necrosis index. It would be bonus help if you provide an image. All help is appreciated. Thank you.
Both EtBr and DAPI could be used for dead cells, that is, when cells are all dead in fixed sample. However, EtBr also stains RNA as well unlike DAPI. In determining live or dead cells in a cell culture, dual staining with PI (propidium iodide) and Hoechst33322 is used to differentiate live or dead cells. PI is permeable to only dying or dead cells while Hoechst33322 is staining every due to its excellent permeability. The resulting dual staining is all cells stained with Hoechst33322 (blue, total No. of cells) and only dying and dead cells stained with PI (red, No. of dead cells). The concentration and incubation time are critical. Refer our paper.
Anticancer Res. 2017, 37(5):2355-2364
Totally agree. An alternative to PI staining is methyl green (MeG), it will behave as PI in terms of cell permeation, so it will only stain damaged cells and it permeates immediately. Besides, its spectral properties are very interesting. It is excited with 633nm light and its emmission peak is around 670 nm, so it gives very clean results despite being very photostable. See attached file.
Please let us know if you solved it.
Best,
Daniel
Article A fast, low cost, and highly efficient fluorescent DNA label...
Dapi stains all nuclei and is membrane permeable along with other fluorochromes such as hoechst.
Ethidium bromide or propedium iodide and I'm sure many other fluorochromes only stain cells that have damaged membranes (i.e. necrotic, late apoptotic dead cells) when unfixed. Several fluorochromes can be used in conjunction to stain cells, including Dapi and ethidium bromide or propidium iodide, as long as they don't interfere with each other.
A good combination would be stains that: 1) differ in their detection (wavelengths) so they don't overlap and 2) have different characteristics to answer your scientific questions. An example is a combination of Dapi (stains all nuclei, live or dead), ethidium bromide or propidium iodide (stains nuclei of only membrane damaged dead cells) and Calcein AM (stains only live cells) (example attached).
All that is required is unfixed cells and a fluorescence microscope with the right filters for the wavelengths of your fluorophores. Fixed cells will give you different results depending on the properties of fixative used. There are many ways to obtain the results you need with fluorescence imaging. It is not very complicated in my case for estimating the percentage of dead cells from total and you can use available options in your lab without stretching your budget.
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