Re: 6 colour sorting

From: Bill Telford (telfordw@box-t.nih.gov)
Date: Tue Oct 23 2001 - 13:56:09 EST


Hi Andy...

We have gotten 6-color to work with FITC / PE / PE-Cy5 / PE-Cy7 off the
argon 488 nm, and Cascade Blue and ELF-97 off a krypton 407 nm laser.
ELF-97 is a fluorogenic alkaline phosphatase substrate sold by Molecular
Probes that can be used for surface immunophenotyping with AP-conjugated
antibodies.  So, we'll use a biotin-conjugated primary, then AP-strepavidin
followed by the ELF-97 substrate.  Although Molecular Probes sells many
different kits for ELF-97 (for tissue sections, suspension cells, RNA,
etc.), we just buy the substrate alone and add it to our cells at 1:20
final concentration.  The cells can then be fixed with formaldehyde if
desired.

ELF-97 is best excited by a UV laser (where it is extremely bright) but
still excites to some extent with a 407 nm beam - it emits in the FITC
range and is still fairly bright with the violet laser.  We then use a 480
LP dichroic to split the Cascade Blue and ELF-97 signals, with a 440/10
filter for Cascade Blue and a standard 530/30 for ELF-97.  There is
virtually no compensation required between Cascade Blue and ELF-97, and
neither are appreciably excited at 488 nm.  Since 407 nm is not the ideal
excitation wavelength for ELF-97, we tend to use it with brighter antigens.
 However, laser power does not seem to be terribly important for ELF-97 -
we get good excitation from 20 to 100 mW (same for UV excitation of ELF-97
as well).

For doing 6-color with a HeNe laser, we have experimented with APC-Alexa
Fluor 680, a tandem also from Molecular Probes.  It is compatible with APC,
but since its emission is similar to Cy5.5, it requires more compensation.
It is brighter than APC-Cy7, though.  We use a 690 LP dichroic to split it
from APC, and a 710/20 BP for detection.  I'm not sure if MP is formally
selling it yet, but they would probably give you some if you asked.

This is an obvious question, but you do have Hamamatsu R3896 PMTs in your
APC / APC-Cy7 detector slots, right?  Some older Vantages might not have
them.

Hope this helps.

Bill Telford
NCI-NIH

  At 10:33 AM 10/22/01 -0700, Andy Johnson wrote:
>
>Dera Flowers
>
>How many people are using 6 colours on a Vantage ?  3 colours off the 488,
>APC plus APC-Cy7 (very weak) with the 633 and Hoechst is fine off the UV.
>Unfortunately I need colours such as cascade blue/yellow and something
>brighter than the APC-Cy7.
>
>Can anybody who uses more than the standard colours give me a suggestion as
>to how to either increase sensitivity or which colours work best.  I also
>have a feeling that it maybe an issue between the Vantage and the MoFlo
>(being more sensitive).  I know all the available fluorochromes out there,
>so what I need to know is which one people have used and got to work.
>
>Would a 120mW from a I-70 laser greatly increase the UV signals, or should
>the 60mW from an enterprise be sufficient ?
>
>Hoping that somebody can help ?
>
>Andy



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