RE: TO-PRO 3 iodide

To all,

A short fixation on ice with freshly prepared buffered formalin or paraformaldehyde alone does not render cells completely permeable to propidium or to-pro-3. You will still be able to dicriminate between cells that were viable or dead before fixation. However, numbers of dead cells will be increased. We label with 1 mM PI or 0.2 mM TO-PRO-3 after fixation. 

Willem Corver


-----Original Message-----
From: Howard Shapiro [mailto:hms@shapirolab.com]
Sent: Tue 26-2-2008 3:30
To: cyto-inbox
Subject: Re: TO-PRO 3 iodide
 
Holly Troche wrote-

> 
> I am labeling MDCK cells with TO-PRO 3 iodide to measure their viability
> in a death assay.  I am currently using 2 ng/mL and staining the cells
> for 5 minutes after which I am washing the dye out (2 wash cycles) with
> a salt buffer. My control cells (presumably alive viable cells) are all
> staining positive for TO-PRO 3. I previously titrated this dye and did
> not find see this phenomenon.  Invitrogen suggests that it maybe because
> I am fixing the cells with 2% paraformaldehyde and this dye is best used
> to dye fixed and permeabilized cells.  Any thoughts? I am considering
> shortening the incubation time period to 1 minute and decreasing the
> concentration further.

I can't even remember how many times I've addressed this issue, but here
we go again.

TO-PRO-3 and other dyes (e.g., propidium) that have double positive
charges, one of which is a quaternary ammonium on the propyl side chain
of the ring nitrogen, do not, as a general rule, enter cells with intact
membranes, but do get into cells with damaged membranes, and therefore
identify "nonviable" cells. Although, for some purposes (e.g., staining
intracellular antigens with fluorescent antibodies or other large
ligands), fixation with formaldehyde or paraformaldehyde does not render
cells sufficiently permeable for staining, such fixation will, in
general, modify the membrane sufficiently for it to let TO-PRO-3 or
propidium in. TO-PRO-3, in particular, may also get into unfixed cells
in early stages of apoptosis.

If you are fixing cells with paraformaldehyde after exposure to TO-PRO-3
and before they are run on the cytometer, there is nothing keeping the
TO-PRO-3 from wandering out of the cells it had initially gotten into
and diluting itself throughout the solution, at which point it will get
into your formerly viable fixed cells. If you are looking for a dye that
will demonstrate nonviable cells but not redistribute after fixation,
your best bet is to use something that will bind covalently to the
nonviable cells. You might also consider TOTO-3, essentially a dimer of
TO-PRO-3; this binds bivalently (although not covalently) to nucleic
acids and therefore has much higher affinity for them than does the
monomeric TO-PRO-3. Dimeric dyes such as TOTO-3 have been reported to
stay on oligonucleotides once bound to them, even when the labeled
oligos are put on gels with other nucleic acid; the TOTO dye might thus
be expected to stay on the cells to which it first gained entry even
after a wash step and fixation. TOTO-3 costs more than TO-PRO-3, but it
does have the same spectral characteristics.

-Howard