Investigation of the Productionof Intracellular Cytokines by T Lymphocytes Using Flow Cytometry – MethodologicalProblems

Summary

The method of assessment of intracellular proteins by means of flow cytometry makes it possible toevaluate the production of different cytokines by a clearly defined cell (sub-population type, stateof cell activation). If the method should become a routine functional test, it must be standardized.This was the objective of our work when, based on data in the literature, we detected all controversialpoints and investigated them experimentally. Quite unequivocally we can recommend onlysodium heparin as an anticoagulation agent when examining whole blood. The paper solvesproblems regarding the selection of mitogens where the marked effect of the use of mitogens on theresult and necessity to compare results obtained under equal conditions was demonstrated. Theauthors tested also the possibility of preserving blood before processing and the selection of suitablecombinations of surface signs and cytokines. When seeking the optimal time for cultivation it isnecessary to make a compromise between the maximum possible production of cytokines (thekinetics of production of different cytokines is moreover different) and the accuracy of measurementbecause detection of the CD4 molecule after a prolonged period of stimulation deteriorates.As the optimum the authors recommend 4.5 hour cultivation with phorbol myristate acetate. Theresults proved a much greater capacity to retain newly formed cytokines in the cell if brefeldin A isused instead of monensine. The outcome of the work is a standard protocol for assessment ofintracellular cytokines.

Key words:
intracellular cytokines – mitogens – protein transport inhibitors.