Flow cytometry

Mon Jul 24 09:38:28 EST 1995

PAIS at wp.path.uab.edu wrote:

>To better understand this problem, it would be necessary to know what 
>kind of background (what specific types of tissue microenvironments show 
>background).  A prblem with Fc mediated background as suggested by the 
>last posting has a characteristic appearance due to the distribution of 
>cells with high FcR levels.  

My cells which I am interested in analysing are splenocytes.  The 
postivity can vary, as the background is concentration dependent.  
The infectious virus is there but I can not see the antigen due to the 
background, at any dilution.   As mentioned prior, I have blocked with 
an Fc block and with normal goat serum to no avail.  

>When you say the "CD antigens" have worked well, exactly what do you 
>mean?  Do you use the same secondary reagent for these as for the 
>murine mAb for the virus?

The CD antigens are CD4,CD8 and B220 (6B2 clone).  All are rat MAb's 
that are directly conjugated to R-PE.  I am double labelling these 
cells and do not get cross reactivity with the rat, as the background 
with the anti-virus Ab alone is the same.  They work well in that 
they are always very clean with at least a log of fluorescent 
separation of positive cells, and the % of CD4, CD8 and B-cells is 
very consistent.

Hope this helps clarify the above questions!


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