RE: 3 colors off 635nm diode

James,

For the 3 colors:
1. APC; 2. read below; 3. APC-Alexa 750 or APC-Cy7

For the middle color, one can use a tandem (APC-Cy5.5, APC-Alexa 700), but a number of
people now use a direct conjugate of either Alexa 680 or 700 with good results. The value
of this latter approach is that, 1. these are available as succinimidyl esters and are
relatively simple for making custom conjugates, 2. they should be more stable than
tandems. In theory, a direct Alexa conjugate should be cheaper to make than a APC-Alexa
tandem, but I'm not sure if this translates into lower pricing of the commercially
available products. 

See the previous Purdue thread : Alexa 680 vs. 700 in polychromatic flow cytometry for
details

http://flowcyt.cyto.purdue.edu/hmarchiv/Current/1099.htm
> _______________________
> Calman Prussin
> Laboratory of Allergic Diseases
> National Institute of Allergy and Infectious Diseases/ NIH
> 
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> ----------
> From: 	James Marvin
> Sent: 	Tuesday, December 6, 2005 10:48 AM
> To:	Cytometry Mailing List
> Subject:	3 colors off 635nm diode
> 
> Hello,
> 
> I was wondering if those of you who have done 3 colors off the red laser 
> could share a little of your experiences.
> 
> 1.Best combination: In the literature I've seen APC, APC-Cy5.5, APC Alexa 
> 680, APC alexa 700, Alexa 700 APC Alexa 750, APC-Cy7.
> 
> As far as commercially available reagents are concerned I would probably 
> end up using APC, and APC-Cy7 together.  What's the best middle 
> fluorochrome with regards to brightness, and compensation.
> 
> 2.  What about Filter sets?  Its seems pretty easy to get custom filters 
> these days so how extensively have you compared different filter 
> combos.  Seems like a common filter for the middle fluorochrome is 
> 700/20.  Seems like you could go a little higher and wider but don't know 
> if you run into comp issues with that.
> 
> I know the spectra viewer is great for this kind of question but some real 
> life experiences might tell a slightly different story.  (then again maybe not)
> 
> Any and all info is welcome.
> 
> Thanks in advance.
> James
> 
>