RE: mCherry on a Calibur ??!!

True, but CMXRosamine has <5% excitation at 488 nm, and gives strong
signal.

Price of reagents/cells/prep notwithstanding, it's worth trying, just
not necessarily worth betting the farm on!

Andrew 

-----Original Message-----
From: Haviland, David L [mailto:David.L.Haviland@uth.tmc.edu] 
Sent: Thursday, August 30, 2007 4:58 AM
To: cyto-inbox
Subject: RE: mCherry on a Calibur ??!!


Rose:

It would depend on the excitation profile.   488 may not be optimal but
if it has, say a
50% excitation at 488 nm you could then detect it.   You might have to
turn up the
voltage of that particular PMT (FL2) but it could be done.     In
contrast, I had a user
who walked in the door with samples stained with Rhodamine (TRITC) not
the Rhodamine 123
that is painfully bright.   Despite warnings, this person waltzed in
thinking the flow
could see everything the fluorescent microscope could.	 Needless to
say, he had to do
without that marker as TRITC is only excited about 1-2% at 488.

So again, I'd check the excitation spectra (profile) and don't base what
can/can't be used on just peak excitation. 

Hope this helps,

David Haviland

-----Original Message-----
From: Rosemary Clarke [mailto:r.z.clarke@dundee.ac.uk]
Sent: Wed 8/29/2007 8:50 AM
To: cyto-inbox
Subject: mCherry on a Calibur ??!!
Dear All,

Now this is going to sound like a very stupid question, so apologies in
advance.

Is it possible to detect mCherry fluorescence on a FACS Calibur, using
488nm excitation and detecting fluorescence at 585nm? I have always
thought not as (in theory at least) mCherry is not excited at 488nm.
However, I have just had a user come to me who thinks he may be getting
a good signal (two log shift from untransfected cells) with what they
believe to be mCherry. However, there could have been a mix up and it
may be dsRed (don't ask!), which would obviously be detectable. They are
in the process of checking and sequencing the constructs but I am now
paranoid that I have got it wrong and mCherry CAN be detected. Has any
one out there in flow-land managed to get a signal from mCherry on a
Calibur using 488nm excitation?

Thanks in advance
Rosie

Dr Rosie Clarke
Division of Cell Biology and Immunology
WTB/MSI
University of Dundee
Dundee
Scotland
UK
Tel: (01382) 385780
E-mail: r.z.clarke@dundee.ac.uk