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John,

As you know, red blood cells are quite unique in that virtually all 
the negatively charged phospholipids (PS, PI, etc) are in the inner 
leaflet of the plasma membrane bilayer (at least in de-nucleated 
human RBCs) and this asymmetry is maintained by a flipase (aka 
translocase). Perturbation of the membrane integrity either 
chemically-physically (oxidative stress, sickling) or by infection 
(e.g., malaria) can lead to a redistribution of PS to the outer 
leaflet. In the case of sickle cell disease, it was shown that an 
increase of PS to as little as 1% on the outer leaflet substantially 
increased the "stickiness" of RBCs and significantly contributes to 
the higher rates of sickle RBC phagocytosis (circulating lifetimes 
for normal RBCs ~ 120 days drop to 11 days for homozygous sickle 
cells).

My main point of bringing all this up is that any fixation protocol 
you might use must be shown to minimally cause redistribution of the 
lipids, PS in particular, across the PM bilayer. The references 
recommended by George, which uses Q-dots, relates to band 3 labeling 
not lipids. As described, cells are absorbed onto a coverslip, 
treated with a crosslinker (DMS) at pH 9.5 then 2% paraformaldehyde, 
blocked with PBS, 3% BSA and 0.2% Tween 20 and so on. Almost any of 
these treatments could perturb the PS/PI distribution and have little 
effect on the band 3 labeling. BSA has two very high affinity sites 
for fatty acids and has been used to extract lyso-PS from the outer 
leaflet.

Labeling PS on the outer surface clearly presents difficulties if you 
wish to preserve physiologically relevant information. The use of any 
detergents, organic solvents, extremes of pH, or even BSA poses 
problems. Using Q-dots has potential advantages such as membrane 
impermeability and photostability. But any probe that is polar and 
large enough, e.g., an Alexa labeled antibody might do as long as you 
can demonstrate that it doesn't create artifacts. You might consider 
using fluoro-gold that can be viewed both by visible light and EM, 
the latter to assure external surface labeling.

Conceivably, there are other possibilities but it would help to know 
more about the specifics of your experiments. You can contact me 
off-list, if you wish. Best of luck.

Mario



>Search the CONFOCAL archive at
>http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
>Dear Farid:
>
>In your confocal-L message of 5 June 2007 (9:13 pm), you noted that 
>you have extensive confocal experience with "FIXED" cells.  That 
>caused my ears to perk up and be curious as to your cell type, and 
>what you do with them.
>
>I am working with red cells that I would like to fix them and label 
>phosphatidylserine-exposed molecules on the surface without the 
>label "leaking" into the cell.  Do you do anything like that with 
>your cells, if not the red cell?  If so, I'd be grateful to hear 
>about it and learn your technique --- IF you've had success.  Or, if 
>you know of anyone who has done what I've described that I'd like to 
>do, I'd appreciate their contact information.
>
>Anyone listening-in who can provide input, I'd be happy to hear from 
>you.  Many thanks.
>
>John Boucher, DVM, PhD
>Email:  [log in to unmask]
>======================================================================

-- 
________________________________________________________________________________
Mario M. Moronne, Ph.D.
ph (510) 528-2400
Fax (510) 528-8076
cell (510) 367-8497

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