I am not aware of it having been used this way previously but the very-long
wavelength membrane probe that we call DiR
1,1'-dioctadecyl-3,3,3',3'-tetramethylindotricarbocyanine iodide ('DiR';
DiIC18(7))
has absorbance and emission well beyond that of hemoglobin. However the dye is
NOT specific for RBC in a whole blood sample but some combination of emission
and scatter may be possible.
[Spectra: 12KB]
It is a structural analog of DiIC18(3), which, under the trademark PKH26, has
been used for cell tracing of reinjected cells by flow cytometry. It should be
possible to excited this dye with the 633 nm He-Ne laser if one has a red
sensitive detector. The quantum yield may not be too high but the
autofluorescence at the emission wavelengths is probably negligible. The
lipophilic dyes are usually nontoxic to cells, I believe.
Because of the good penetration of light beyond 700 nm through skin it may also
be possible to see the DiR-labeled red blood cells in vivo given quite special
imaging equipment if there are enough of them.
As an alternative, the shorter wavelength "DiD" analog
http://www.probes.com/servlets/product?region=USA&item=307
(which is spectrally similar to Cy5 and Alexa Fluor 647 dyes) has better
absorbance at 633 nm and its emission is at shorter wavelengths.
[Spectra: 12KB]
It can be detected well by regular detectors at these wavelength but is somewhat
more likely to FRET to the hemoglobin.
I am not aware of this having been tried either for this application.
A possible protocol for labeling is here:
http://www.probes.com/servlets/product?region=USA&item=22887
"Richard K. Meister" wrote:
> Hello, everyone!
>
> I have a colleague who wants to draw blood from a dog, label the
> erythrocytes with a permanent (or semi-permanent) fluorescent tag, reinfuse
> the cells into the dog and later (15 minutes) draw blood from the dog again
> and run the RBCs on the flow cytometer to determine the recovery of
> previously-labeled cells. The proposal sounded simple enough to me until I
> started to look for the required stain or monoclonal antibody that would
> specifically and uniquely stain the RBCs in a sample of whole blood.
>
> I haven't found anything yet, so any ideas you might have would be welcomed.
>
> Thanks in advance.
>
> Rick Meister
>
> * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * *
> * Richard K. Meister Email: meister.1@osu.edu *
> * The Ohio State University Voice: (614) 292-9716 *
> * Dept. of Veterinary Biosciences FAX: (614) 292-6473 *
> * Cytometry Instrumentation Lab *
> * 1925 Coffey Road *
> * Columbus, OH 43210 U.S.A. *
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