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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