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Whole Blood Protocol for Analysis of Intracellular Cytokines by Flow Cytometry
For the staining of intracellular antigens in whole blood using directly conjugated antibodies.
This is a rapid and simple approach to the analysis of intracellular cytokines in whole blood. It permits the analysis of small samples and avoids generating artefacts due to the separation of peripheral blood cells by density gradient centrifugation. All blood samples must be collected into heparin anticoagulant. EDTA interferes with the cell stimulation process and therefore must be avoided.
The detection of intracellular antigens requires a cell permeabilization step prior to staining. The method described below has been found to provide excellent results in our hands; however other permeabilization techniques have been published and may also be successfully used in this application.
This method provides a general procedure for use with the majority of Bio-Rad reagents. In some cases specific recommendations are provided on product datasheets, and these methods should always be used in conjunction with the product and batch specific information provided with each vial. A certain level of technical skill and immunological knowledge is required for the successful design and implementation of these techniques; these are guidelines only and may need to be adjusted for particular applications.
Note: Resting cells often require stimulation in vitro prior to the detection of intracellular cytokines.
Sewell, W.A. et al. (1997). Determination of intracellular cytokines by flow cytometry following whole-blood culture. J. Imm. Meths. 209: 67-74
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