Hello everybody,
I was trying to monitoring dead cell in seaweed tissues (in this case of Gelidium). I was trying with Erythrosine and Trypan Blue but do not stain very well dead cells...and even fresh tissue (as a negative control for both) is stained in some cases. Concentration, time of incubation, time of washing, etc...were some of the factors that I have tested but the results are the same. Any recommendation? These dyes have been used to assess viability of microalgae, filamentosus seaweed (incluiding gametophytes) and seaweeds with 2 o 1 layer of cell...so the intercelular matrix could be a serious problem in my case as i m working with Gelidium. I have no tried yet with Evans blue.
Many thanks
Not sure how well it would work with macroalgae, but for microalgae (and apparently zooplankton eggs), TO-PRO-1 iodide staining works very well for distinguishing live cells from dead ones.
Hello, using epifluorescene microscopy is the better one. Since algal have a strong autofluororescent, I think you check first weather your algal dong have autofluororecent and you can choose the probe, which may be suitable for you. I play with algal.
1) Calcien Violet AM (C34858) combined with TO-PRO-3 (T3605): Calcein violet AM works the same way as Calcein green AM, where it will only fluoresce in live cells. This dye needs excited at 405nm (violet emission), so you will have to check if your system has that wavelength. Emission is around 450nm. You will have to check your cells to see if they autofluoresce at 450nm. TO-PRO-3 is similar to Ethidium homodimer in that it is non-cell-permeant. It will enter dead or compromised cells and bind to nucleic acids, at which point it will fluoresce far red, similar to Cy5 (excitation peak at 642 nm, emission at 661), so you will need to check if your system has a filter for that and if your cells autofluoresce in far red (few cell types do).
2) Hoechst 33342 (H1399 or H3570) combined with TO-PRO-3 (T3605): Instead of a live cell indicator like Calcein green or violet, you can label all cell nuclei with Hoechst. It is UV-excited and blue-emitting and labels with the same spectrum and mechanism as DAPI, but is more cell-permeant. This can be combined with TO-PRO-3. Thus, all cells will label with Hoechst, and only dead cells with TO-PRO-3 (and thus any cells that are negative for TO-PRO-3 can be deemed as “live”).
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