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I'm afraid that the neurotrace gels are rather a dead-end as far as
labeling cells in culture are concerned. The method I found to work best
for this purpose was to complex DiI, DiO, DiD etc with BSA and then add the
aqueous complex to the culture. If you download the protocol for BODIPY
dye labeled sphingolipids from the Life Technologies website, you will find
it contains a protocol for making BSA complexes of fluorescent ceramides.
Use this protocol, substituting DiI etc for the fluorescent ceramide. The
protocol for preparation of the ceramide:BSA complex calls for an initial 1
mM dye solution in chloroform:ethanol. With DiI, you can dispense with the
chloroform as it is soluble directly in ethanol at 1 mM. Also, the
protocol calls for using the dye:BSA complex at a 1:100 working dilution.
With DiI, I would use a 1:1000 working dilution as a starting point
instead. The dye:BSA complex in water or PBS is quite stable and can be
stored and re-used for years in my experience.
Iain
Iain Johnson Consulting
Eugene, OR
On Thu, Jun 6, 2013 at 2:32 PM, Craig Brideau <
[hidden email]>wrote:
> *****
> To join, leave or search the confocal microscopy listserv, go to:
>
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy> *****
>
> Hello all, I recently got my hands on a Neurotrace Gel kit from Life Tech
> (Invitrogen) and want to use it in a non-standard way. The kit I have is
> the DiO, DiL, and DiD combo. I want to use one or two of these dyes to
> label cultured neurons in a dish. The problem is that the Gels are
> normally meant for tissue, and you just sort-of smear the gell directly
> onto a tissue slice or the like. The cells we have are just cultured on
> the bottom of a dish in some PBS. Any thoughts as to how one might get the
> Water-resistant Gel containing Di into the cells? I was thinking of trying
> an emulsion into the PBS but I'm not sure that would work.
>
> Thoughts?
>
> Craig
>
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