Oxidative burst assay

Hi Flowers,

I am a beginner in this field so I was hoping that someone might be able
to offer some advice! 

I have been working on an opsonophagocytosis assay which measures the
complement mediated antibody dependent uptake of PKH26 stained bacteria
into HL60 cells. I am trying to incorporate DHR123 into this assay to
measure oxidative burst response. To check that measuring these two
parameters simultaneously was not effecting the assay results I have run
it with a variety of sera from different species and have looked at the
responses in a variety of ways. I have done an assay with DHR123 on its
own with unstained bacteria, an assay measuring just the uptake of
PKH-26 stained bacteria, and an assay measuring both DHR123 and PKH26
stained bacteria uptake.  The assay with DHR123 on its own with
unstained bacteria, and the assay with DHR123 and PKH26 stained bacteria
are not correlating. There are sera with much larger DHR 123 values in
the combined assay, whereas the rest appear fairly comparable. Following
an assay repeat the same thing happened but with different sera from the
same set.  However the assay measuring just PKH-26 stained bacteria
uptake correlates very well with the assay measuring both parameters.
Could any one offer any indication as to why this could be happening??
Is it something to do with the stains interacting or could it be the
cytometer settings (we have an FC500)? Any suggests would be
appreciated.

Thanks in advance

 

Charlotte Brookes

 

 

Research Scientist

Meningococcal Vaccine Group

Health Protection Agency,

Porton Down,

Salisbury,

SP4 OJG.

 



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