Doesn't PI meet your criteria?

a lot of esterase-activated substrates, ion sensors look like similar. for me the question is little bit straitforward, all in-cell fluo substrate/sensor must behave this way. search CellTracker Probes from Thermo Fischer for example. (i suppose you want to stay your probe inside the cell).

thanks for the answers so far.

Currently i'm not quite sure if PI is suitable for my purposes. I'll work with live cells with intact membranes. If I bring PI inside the cell, Is PI able to cross the nuclear membrane if the cell is intact and consequently show fluorescence?

@Attila:

I was searching a lot for celltracker probes but I couldn't find any matching my criteria:

Either the Celltracker is membrane-impermeant but shows fluorscence independt of it's location or the Celltracker gains fluorescence inside the cytosol (cleavage by esterases e.g.) but is also cell-permeant.

I couldn't find any which is cell-impermeant and shows fluorescence only if it's inside the cell (cytosol preferentially).

Kind regards

Robert

PI stains dead cell only. usually probes are taken up by cells and that is why do not signal outside the cell. H33342 stains live nuclei.

" CellTracker Orange CMRA also requires esterase cleavage to activate its fluorescence. CellTracker Orange CMRA is a rhodol-based fluorophore with an overall net charge at neutral pH that is expected to be negative, in contrast with the overall net positive charge of the tetramethylrhodamine-based CellTracker Orange CMTMR. Thus, unlike CellTracker Orange CMTMR, CellTracker Orange CMRA should remain primarily in the cytoplasm instead of being sequestered inside actively respiring mitochondria."

@Attila

Tank you for your answers.

But my main problem still exists: I need a cell-impermeant dye which cannot passivly and freely cross membranes.

When I m not mistaken, the depicted CellTracker Orange is able to cross membranes freely?

Thanks again and kind regards

Hi,

according to the description, it is inactive (no fluo), goes across membrane, then activated by esterases (non-specific); after that, remains in the cytosol mostly (not comes out from cells). That is not personal experience. That's all. If you feel unconfident, try to ask a sample. Earlier Mol Probes was not a so "generous" company, Thermo looks different for me. Good luck! 

Thanks again,

According to the description i cannot use it because it crosses the membrane freely (prior to the esterase cleavage).

Do you need some active transport for the uptake (ie your target is that)?

may be NR12S can work? it stains only membrane (outer layer can be switched off by dithionite upon spectroscopy/imaging)

I would like to transport the dye actively into the cell by a carrier system. That s why i need a dye which cannot freely cross the membrane. In addition, my carriersystem is fluorescnece labled as well and for analysis via fluorescence cytometer i would like to have a cell laabeling dye which becomes active if it s inside the cell --> thereby i could distinguish between cells which are effectivly targeted by the carrier system and a release of the dye occured

In such cases, rather a fluo-labelled substrate of the carrier system is appropiate (there can be more, of course, i mean substrates, even fluo ones). Your case is extra in that aspect, that you need it non-fluo form. esterase activation in cell is still can be work, however, a carrier can be work at reverse mode too. Good luck, again.

It sounds like you are looking for an endocytosis tracking probe? (Membrane impermeable dyes cannot stain organelle membranes. For this, you need fluorescent proteins or enzyme tags.) There are pH sensitive probes out there that can change its spectra once the endosome pH lowers, but you will miss the earlier part of the process. You can use bright and stable probes, like quantum dots, then add the quencher outside the cell to 'turn off' the fluorescence outside the cell.