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Additional resources for Cytokines and Colony Stimulating Factors: Methods and Protocols
6. , Zangerle, P. , Fassotte, M. , et al. (1992) Direct stimulation of cytokines (IL-1`, TNF-a, IL-6, IL-2, IFN-a, and GM-CSF) in whole blood. I. Comparison with isolated PBMC stimulation. Cytokine 4, 239–248. 7. , and Klüter, H. (2001) Ex vivo induction of cytokine mRNA expression in human blood samples. J. Immunol. Methods 249, 63–71. 8. Maino, V. , Ruitenberg, J. , and Suni, M. A. (1996) Flow cytometric method for analysis of cytokine expression in clinical samples. Clin. Immunol. 16, 95–98.
1-fold is seen (10). Isotype-matched antibodies display the inherent staining background for a given ﬂuorescent antibody, but they fail to cover the enhancement of nonspeciﬁc staining induced by the procedure of stimulation, permeabilization, and ﬁxation (Fig. 3). Therefore, the puriﬁed antibody blocking control should be preferred to each sample. Nonstimulated cells are also an unreliable negative control because cytokine positive cells can be found without in vitro stimulation. Moreover, the augmentation of nonspeciﬁc staining during stimulation cannot be detected in nonstimulated cells (Fig.
Beyond the information already provided, the author would like to share a few additional comments for a successful performance of the assay: 1. The inability to detect a cytokine signal may be the result of insufficient permeabilization or inactive BFA or LPS solutions. Incorrect storage conditions may inactivate LPS or BFA solutions. It is imperative that correct dilutions and concentrations for solutions like FACS Permeabilizing, BFA, or LPS are used. Blood drawn in other anticoagulants than sodium heparin should not be used.