sorting murine eosinophils on FACSAria II

Howard Shapiro hms at shapirolab.com
Wed Dec 3 19:49:58 EST 2008


Frank Battye responded to Monica DeLay:
> In ancient times we used to sort mouse Eos from neutrophils using 
> their higher blue "autofluorescence" as excited by UV laser.	Your 
> Aria could do that if it has a UV laser.   Don't know if 405nm would 
> do it for you if that's all you have.  The fluorescence comes from the 
> cells' NAD(P)H system.  See, for example, Vadas et. al. Blood 61, 
> p1232-41 1983 or Watt et. al. J Histochem Cytochem 28 p934-46 1980.  
>
>> We have a customer that would like to sort eos from mice without 
>> staining them.  They
>>
>> will be enriched (~90% pure) using MACS but they want to purify them 
>> from the
>>
>> Neutrophils.  We used to sort for eos on the Vantage using 
>> depolarized light scatter.  I
>>
>> have 2 questions:
>>
>> 1.  Has anyone found a way to do this on the FACSAria?
>>
>> 2.  If not, is there a way to negatively select for eos.  I've seen 
>> using CD16 as eos are
>>
>> negative for this (at least in human). Is this a standard protocol in 
>> mice?
>>
Actually, eosinophil autofluorescence is pretty broadband. In addition 
to the pyridine nucleotide fluorescence Frank mentions, you also see 
green to yellow autofluorescence probably largely due to flavin 
nucleotides; that excites well at 457 nm but also at 488 nm. It's pretty 
dim; we estimated it as a few thousand fluorescein equivalents.

The other thing about eosinophils is that they express CD4, again, at 
low levels, probably lower than monocytes, but typically detectable on a 
CD4 vs. side scatter plot (eos have side scatter signals at least as big 
as those from neutrophils).

You wouldn't be able to do depolarized scatter because the fiber optics 
between the collection lens and the octagons/trigons in an Aria don't 
preserve the polarization of collected light.

-Howard




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