I am measuring ROS production in SH-SY5Y cells using DHE (emits red fluorescence) and I'd like to concurrently measure the level of cell death in those same samples.
Naturally therefore using PI staining is not an option. Since I am concurrently using Hoechst 33342 to assess cell density, I can also therefore not use a marker for cell death that emits a blue fluorescence.
Is anybody aware of a marker that I might be able to use alongside the DHE (red) and Hoechst (blue), say for instance, something that emits a green fluorescence?
Thanks in advance!
Dear Thomas,
You can use a SYBR green based dye that will emit in the FITC channel. An example of such a kit is CellTox Green from Promega. It works in the same way as the PI stain: dead cells will have a permeable membrane and take up the dye.
https://be.promega.com/products/cell-health-and-metabolism/cytotoxicity-assays/real-time-cytotoxicity-assay/celltox-green-cytotoxicity-assay/
Kind regards,
Good luck!
Bjorn
In addition to the answer above, another option is the use of annexin V, which is often conjugated to a green fluorophore, such as FITC or alexa fluor 488. Annexing V binding is a marker of apoptosis. More specifically, it detects re-organization of cell membrane lipids, which occurs early during apoptosis. Thus, it might be more sensitive for detecting cell death if, under your experimental conditions, the cells have only begun to initiate apoptosis and have not yet lysed fully, which would be detected by PI and similar cell impermeable dyes.
https://www.thermofisher.com/order/catalog/product/A13201
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