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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