Intracellular staining for TNFa

catherine haluszczak halusz at hotmail.com
Wed Sep 27 14:16:27 EST 2000


Dear flow experts,

We have been doing intracellular staining using BD reagents and antibodies
for about one year and have recently encountered a problem we can't resolve.
  In our study on controls vs pregnant samples we usually run a BFA only
group and have started to see problems with all CD3 PERCp or CyChrome
positive cells being also 100% positive for TNFa APC.  The PMA-Ionomycin
group will also be 100% positive for TNFa with a subgroup being double
positive for IL2-FITC. IFNg vs IL2 dot plots look normal. We usually add all
three cytokine antibodies together as a mixture. This will occur on all
subjects on a given day then the next test, a day or two later, using the
same reagents BFA samples will be negative and the normal profiles for all
three cytokines will be normal.

To me this does not make biological sense. What could be different
technically to cause erratic results? We tried to do a blocking experiment,
ie cold TNFa then TNFa APC labelling with out the other cytokines in the
mix, like IL2-FITC and IFNg PE, there was no TNF staining with or without
the cold block.
Help.

Catherine Haluszczak
Research Specialist
Children's Hospital
Division of Immunogenetics
Pittsburgh, PA



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