James What probably is happening is that at high differential the core stream is becoming very wide relative to the cell diameter. Thus cells can come through side by side (or some partial side by side). Thus negative cells can be sorted with the positives by hiding with them since the sorter can not distinguish (by fluorescence) a positive alone from a positive plus a negative except by some type of doublet discrimination which will might fail (are you doing doublet discrimintation using FSC/pulse width/integrated FSC). Thus the bottom line is it is always critical to try to keep the differential as low as possible. On my MoFlo I try to not go more than about 0.3-0.4. If the cells are too dilute they will either have to pay you for extra time orf concentrate the cells. Larry At 11:57 AM 4/12/2002 -0600, you wrote: >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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