Good day, Within the last couple of weeks we have been having purity issues with a MoFlo. When we reanalyze some of the samples we get a mixture of cells. When this happen we recheck the drop delay and it is exactly where it should be. I am stumped on what the problem could be other than electronic problems. In my attempt to rule out one by one all of the foreseeable problems, one thing that popped into my mind was that we often have people come in and have no clue, or, are way off on what they think there sample concentration is. So often times I run the samples with a high differential. Sometimes as high as 1 psi above sheath pressure. I'm pretty new to flow cytometry but believe that the wider the sample core (high differential) the less hydrodynamic focusing takes place and therefore the cells will not be perfectly aligned. So basically we are not getting a true picture of what the fluorescence is of a particular cell. So then when we reanalyze the cells at a lower differential the cells have a different, more accurate fluorescent pattern. So my question is. Is this a legitimate concern? Are you strict with clients to have their samples at an optimal concentration in order to keep differential low? Any other suggestion on what the cause of impure sorting can be? I know that last one is an endless black hole but I thought I'd try it out anyway. I will mention that in one particular case there was no compensation needed, instrument setup was optimized just like every other day, staining in controls and sample looked great, and drop delay was right-on. Thanx a ton marv
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