RE: Baclight live/dead staining

From: Gerhard Nebe-von-Caron (Gerhard.Nebe-von-Caron@Unilever.com)
Date: Tue Mar 06 2001 - 06:10:15 EST


At lower concentrations the SYTO9 can be actively removed from the cell by the
proton antiport pumps as the efflux exceeds the influx.
Another factor that influences your count (apart from the actual counting
technique used) is the selected trigger or threshold. On the Beckman Coulter
Elite we use multichannel triggering to see particles either based on their red
or green or light scatter signal. Most instruments only allow you one
threshold. If for interference or sensitivity reasons you might not be able to
use light scatter you could try triggering on FL2 as both dyes have
considerable signal overlap into the orange (you could even up the voltage on
that channel).

Hope this helps
Gerhard



-----Original Message-----
From:	Paul Thomson [SMTP:paul_tho@antdiv.gov.au]
Sent:	Monday, March 05, 2001 3:09 AM
To:	Cytometry Mailing List
Subject:	Baclight live/dead staining


Dear All,
I'm trying to develop a reliable method to count Baclight stained live/dead
marine bacteria using a
FacsScan (PI for dead, CYTO 9 for live).  The problem I'm experiencing is
this.  While I can differentiate between live and dead populations (using
FL1 vs FL3 channels), the counts I get on the FacScan account for <50% of
the numbers I get using fluorescent microscopy.  I suspect that some of the
less fluorescent cells I count on the microscope do not stain up well at
the stain concentrations
I'm using for the flow.  When I increase flow stain concentations, I
suspect they are lost in the noise created by non-specific fluorescence
caused by unbound stain.

Any advice would be greatly appreciated.

Cheers,
Paul



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