I've got specimens with a small amount of cells. Hoechst is not good in this case, cause it gives me information not only about viable cells, but also about all of the "rubbish" in a specimen. I would like to avoid it and get to know which elements are really cells. Please, help.
I would recommend the LIVE/DEAD Kit (Molecular Probres) which is based, I believe, on Calcein-AM + ethidium homodimer-2. Calceim-AM is a non-fluorescent cell permeable ester that once enter inside live cells gets cleaveaged by endogenous estereases and becomes non permeable and fluorescent in the green channel. EtHD works as Hoechst, staining dead cells but it shows red fluorescence.
I used this two as a homemade kit some time ago and it worked fine for live/dead staining.
Calcein AM is a very good dye for identifying live cells. It is non-fluorescent until it enters lives cells and will leak out of dead cells. Note that, depending on the cell type, it will only be retained for 2-6 hours as it is pumped out by anion drug transporters.
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