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 _________________________________________________________________________ Get Your Private, Free E-mail from MSN Hotmail at http://www.hotmail.com. Share information about yourself, create your own public profile at http://profiles.msn.com.
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