FRET using FACScalibur

Janos, Szollosi szollo at jaguar.unideb.hu
Tue Feb 15 15:16:06 EST 2005


Dear Ankita,

The paper bleow describes how to perform FRET measurements on 
FACScalibur.  It also offers autofluorescence correction on a cell by cell 
basis.

Sebestyén, Z., Nagy, P., Horváth, G., Vámosi, G., Debets, R.,  Gratama, J. 
W., Alexander, D.R., Szöllösi, J: Improving flow cytometric energy transfer 
measurements on a commercial flow cytometer using cell-by-cell correction 
for autofluorescence.
Cytometry, 2002. 48, 124-135.


However in your case the situation can be a bit more complicated.  Can I 
assume from your short description that the ligands and the receptors are 
on two separate cells?	If yes, how would you keep the cells together in 
the flow system? Even if you succeed only percent of the receptors will 
interact with the ligands [for sterical reasons] so the FRET signal will be 
overshadowed by fluorescence coming from molecules not participating in the 
FRET process.

For this purpose microscopic measurements could be a better solution.

Do not hesitate to write me directly with more specific questions!

Regards

Janos



At 11:04 PM 2/11/2005, Garg, Ankita wrote:
>Hi all,
>I have to perform receptor-ligand interaction on 2  cell .I want do it 
>using FRET, but am
>not sure how can I do it with flowcytometr . I work on FACScalibur with 2 
>lasers. Please
>help me out.
>thanks in advance
>ankita
>
>-----Original Message-----
>From: Nebe-Von-Caron, G [mailto:g.nebe-von-caron at unipath.com]
>Sent: Thursday, February 10, 2005 12:40 PM
>To: cyto-inbox
>Subject: RE: Sorter sterilization
>
>
>According to a beer advert there are beers that reach parts not reached
>by others, but I can not see that to be the case for disinfectants.
>Hypochlorite and alcohol is as potent as you can get. You are more
>likely to miss some crucial part in the cleaning routine (valve, air
>trap...). If you sort below 40PSI I recommend Millifill-GV disposable
>online filter(2 in parallel) close to the flow cell. That should stop
>anything from that end.
>
>Good luck
>
>Gerhard
>
>
>-----Original Message-----
>From: Barren, Phil [mailto:BarrenP at MedImmune.com]
>Sent: 04 February 2005 21:35
>To: cyto-inbox
>Subject: Sorter sterilization
>
>
>FLOWers:
>
>Can someone tell me the concentration of hydrogen peroxide they use in
>sterilization of a cell sorter.
>
>  I have tried ETOH and Bleach ( various concentrations), but my low
>level contamination continues, so I want to try peroxide.
>
>Any wisdom would be appreciated
>
>Tks
>
>Pb

Janos Szollosi
Department of Biophysics and Cell Biology
Faculty of Medicine
Medical and Health Science Center
University of Debrecen
P.O.Box 39, Nagyerdei krt. 98
H-4012 Debrecen
Hungary
Phone/Fax:  (36) (52) 412-623
E-mail:  szollo at jaguar.dote.hu





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