DAPI and APC on 2nd laser lines

Witherspoon, Sam sw11527 at glaxowellcome.com
Wed Apr 19 07:24:32 EST 2000


Please accept my apologies for having APC on the brain
	The reference below was published as using Cy5,  not APC.
Still, my desire is to use APC in my application if possible.

	Thanks,
		Sam Witherspoon

> -----Original Message-----
> From: Witherspoon, Sam
> Sent: Tuesday, April 18, 2000 7:50 PM
> To:	'Cytometry Mailing List'
> Subject:	DAPI and APC on 2nd laser lines
>
> Hi Group
>	I'm trying to collect DAPI and APC signals on the second (and third)
> laser lines of a FACStar Plus in a manner very similar to that described
> by Moore et al in:
>	      Cytometry (32) 1: 57-65 , May 1998.  The method describes the
> collection of DAPI and  [Sam]  (was Cy5 not APC) APC fluorescence from
> cells excited by collinear beams of a mixed gas laser for UV (350-356 nm)
> and a HeNe laser (633 nm).
>
> In this technical note,  the filters adjacent to the relevant PMT's are
> described (660DF20 for  [Sam]  Cy5, not APC )  and 450DF20 for DAPI), but
> the splitter between the two PMT's is not mentioned.
>	I have looked around and cannot find a reference as to the "proper
> thing to do" in this case.
>
>	The DAPI signal is very bright... should I reflect it and filter the
> APC fluorescence or should I filter the DAPI and reflect the APC?
>
> Is there a general rule that I could apply based on wavelength regardless
> of "brightness"?
>
>	Thanks in Advance,
>
>	I look forward to your input.
>
>	Cheers,
>		Sam
> Sam Witherspoon
> sw11527 at glaxowellcome.com
>
> Dept. of Receptor Biochemistry  Tel. 919-483-3078
> Glaxo Wellcome R&D		  Page 919-857-7768
> 5 Moore Dr.			  Fax  919-483-0585
> RTP, NC  27709
>




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