Molecular Probes Live/Dead Kit

Howard Shapiro hms at
Tue Jun 15 20:55:23 EST 2004

Haywood Pyle wrote:

>Is anyone using the Molecular Probes Live/Dead, Viability/Cytotoxicity 
>Kit?  We are trying to put into place a viability procedure that measures 
>metabolic activity as well as membrane integrity.  This would replace our 
>usual trypan blue testing.  We have tried this kit with a FACS Calibur and 
>have problems with dual expressing cells which Molecular Probes technical 
>assistance has told me should not exist.  I suspected compensation issues 
>but no amount of compensation resolves the problem.  If someone out there 
>has any experience with this kit I would love to ask some questions.

I have not used the kit, nor have I used its components (calcein-AM and 
ethidium homodimer) together or separately. However, since the issue keeps 
coming up, I feel compelled to take issue with Haywood's description of the 
procedure as measuring "metabolic activity" as well as membrane integrity. 
Molecular Probes' describes their kit, more accurately, as indicating 
esterase activity and membrane integrity.

Ethidium homodimer is, as advertised, a dimer of ethidium. Ethidium itself, 
although often used in "dye exclusion" tests of membrane integrity, bears 
only a single positive charge, and can be taken up by cells with intact 
membranes, from which it is usually pumped out. Propidium, which differs 
from ethidium in carrying a positively charged quaternary ammonium group on 
the propyl substituent of the ring nitrogen, is excluded from cells with 
intact membranes, as are ethidium homodimer, Sytox green, and dyes of the 
YO-PRO and YOYO series, all made by Molecular Probes and all bearing at 
least two positive charges.

Calcein is a fluorescein derivative, with more carboxyl groups than 
fluorescein; its acetomethoxy ester (calcein-AM) is, like the diacetyl 
ester of fluorescein (universally, if not strictly accurately, referred to 
as fluorescein diacetate, or FDA) nonfluorescent, uncharged, and 
lipophilic. By virtue of their lipophilicity, both calcein-AM and FDA can 
easily cross intact cell membranes; once inside cells, they are hydrolyzed 
by nonspecific esterases, yielding fluorescent anionic compounds which 
leave cells relatively slowly (minutes in the case of FDA, possibly weeks 
in the case of calcein-AM). It is known that intracellular fluorescein 
accumulation in cells exposed to FDA is not energy-dependent, proceeding 
approximately as rapidly in cells treated with inhibitors of energy 
metabolism as in control cells, establishing that FDA enters cells 
primarily by diffusion through the lipid bilayer. There is some evidence 
that efflux of FDA from cells with intact membranes is energy dependent. 
However, as typically used, FDA discriminates between cells with intact 
membranes, which retain the dye, and cells with damaged membranes, from 
which dye leaks out much more rapidly. Thus, one gets basically the same 
information from FDA or calcein retention and from exclusion of dyes such 
as ethidium homodimer, propidium, Sytox green, etc - i.e., that the 
membrane is intact. Even cells with damaged membranes may contain active 
esterases; they just don't retain the products. "Metabolic activity" in the 
sense in which it is usually understood is better demonstrated using dyes 
responsive to energy metabolism, for example, indicators of mitochondrial 
membrane potential.

I can't speak from experience about calcein-AM and ethidium homodimer, but 
there are situations in which bacterial and eukaryotic cells can show 
simultaneous staining by FDA and propidium or TO-PRO-3, and I suspect that 
what is going on in at least some of these cases is that the nucleic acid 
dye (e.g., propidium) is getting across the intact membrane with the aid of 
a transporter. However, instrument artifacts could also explain at least 
some of the dual positive cells.

The advantage of a two-dye combination such as calcein-AM/ethidium 
homodimer or FDA/propidium over a single dye is that it should show you all 
of the cells; if you use either dye alone, relying on a scatter threshold 
or gate to define "cells", you may not get as accurate a count of the 
unstained population.


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