I was thinking of using Propridium Iodide as a live/dead nuclear stain. The trouble is that I am already tied in to using pHrodoRed labeled Abeta peptides as a phagocytotic marker. I do not think that the PI can be used with the pHrodoRed.
Here are the spectra characteristics that I have found so far:
Note: PI is excited at 488 nm and, with a relatively large Stokes shift, emits at a maximum wavelength of 617 nm.
Once the dye is bound, its fluorescence is enhanced 20- to 30-fold, the fluorescence excitation maximum is shifted ~30–40 nm to the red and the fluorescence emission maximum is shifted ~15 nm to the blue, resulting in an excitation maximum at 535 nm and fluorescence emission maximum at 617 nm.
The pHrodo™ red dye (pHrodo RED # P36600, life technologies); has excitation and emission maxima of approximately 566 nm and 590 nm and can be detected with standard TRITC (tetramethylrhodamine) or Alexa Fluor® 555 filters.
Does anyone have a suggestion as to how I can do a live dead stain and also see phagocytotic uptake of my pHrodoRed probe?
Thanks,
Neal from sunny Seattle
Neal,
there are many dyes/stains that are available, but which will work with your system depends on a few things:
- What other dyes or stains are you using (you've mentioned this)
- What lasers or excitation wavelengths are available (you haven't mentioned this)
- What detection channels are available (you've not mentioned this either.
It 'sounds like' you might have a 488 laser and possibly a 565 laser. is that right?
Does the system you are using have a UV laser? (around 355 nm)
Does your system have a violet laser? (around 405 nm?)
Does your system have adjustable band pass filters? Or are they set?
There are a fer Hoescht Dyes that may work for you but it depends on the lasers and detectors you have.
Can you get back to me with more information?
Hi Neal, You could use any of the fixable viability dyes (Thermo calls them Live/Dead and BioLegend calls them Zombie). They have all sorts of colors that could fit your needs.
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