[Cytometry] Out of scale signal and negative population

Will Schott Will.Schott at jax.org
Tue May 10 11:16:13 EDT 2011


This is due the the baseline subtraction that the machine does. If there is
a lot of fluorescence between events, your negatives will be below zero.
This can be due either to unbound antibody from insufficient washing, or
from binding of the antibody to large amounts of cellular debris. If it is
from insufficient washing, additional washing will help. If it is from
debris then running at a lower speed helps as does lowering the thrshold to
the point where the debris is also counted as events. We see this with
really bright GFP samples with lots of debris as well as with samples where
the antibody was not washed out. Some channels are much more sensitive to
this than others. 
-will 


On 5/9/11 12:05 PM, "COSMA Antonio 224446" <antonio.cosma at cea.fr> wrote:

> Dear All,
> 
> During some titration experiments, we noticed that in samples where the
> positive signal was out of scale the negative population moved to the minimum
> value of the scale below zero. Decreasing the amount of Ab was bringing the
> positive population in scale and at the same time the negative population
> increased the autofluorescence and came back in scale, too.
> During the setting negative cells were placed on the first decade of the
> scale.
> 
> We observed this phenomenon on a Fortessa equipped with DIVA software.
> 
> Any idea...
> 
> Best
> 
> Antonio Cosma  
> 
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William Schott
Flow Cytometrist
The Jackson Laboratory
600 Main St.
Bar Harbor, ME 04609
Phone: (207) 288-6192







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