PMID: 9448035Feb 3, 1998Paper

Determination of intracellular cytokines by flow-cytometry following whole-blood culture

Journal of Immunological Methods
W A SewellJ Farrant

Abstract

Various methods have been reported for measuring intracellular cytokines in peripheral blood mononuclear cells isolated by density-gradient centrifugation. In this report, we describe a whole-blood method for the determination of intracellular cytokines (IFN-gamma, TNF-alpha and IL-2) that uses small-volume (500 microl) blood samples. Directly conjugated anti-cytokine antibodies and commercial cell membrane fixation and permeabilisation reagents were used. Blood was cultured in a 1:3 dilution with a combination of PMA and ionomycin to reveal the cytokine synthetic potential of each cell, together with monensin to increase the sensitivity by retaining cytokines within the cell to detectable levels. The optimum concentrations of PMA (10 ng/ml (16.2 nmol/l)), ionomycin (2 micromol/l) and monensin (3 micromol/l) were determined. Kinetic studies showed maximal cytokine expression after 2 h of culture for TNF-alpha and IFN-gamma and 4 h for IL-2. Assessment of TNF-alpha and IFN-gamma production within the CD4 and CD8 lymphocytes from 10 normal volunteers showed that considerably more CD8 + than CD4 + cells produced IFN-gamma. This technique could be used by routine immunology laboratories and will be of use in studies to determine wh...Continue Reading

References

Sep 1, 1992·Blood Reviews·R ThorpeA R Mire-Sluis
Feb 26, 1993·Journal of Immunological Methods·T JungC Rieger
Jul 1, 1996·Immunology Today·U SchauerA Frew

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Citations

Apr 30, 2003·Cellular Immunology·Fabiola AtzeniFranco Capsoni
Sep 15, 2000·Journal of Immunological Methods·P PalaP J Openshaw
Feb 28, 2001·Journal of Immunological Methods·C HärtelH Klüter
Nov 11, 1999·Journal of Immunological Methods·H TayebiE Robinet
Jun 18, 2002·Clinical and Experimental Immunology·R H BuckJ E Dugle
Feb 7, 2009·Journal of Leukocyte Biology·Gregory B PottLeland Shapiro
Jul 13, 2004·Clinical Pharmacokinetics·Christine E Staatz, Susan E Tett
Jun 11, 1999·Clinical and Experimental Immunology·W A SewellJ Farrant
Jan 18, 2006·The Journal of Investigative Dermatology·Claire PelletCéleste Lebbé
Dec 22, 2004·BJU International·John D BeattyChristopher W Ogden
Apr 19, 2006·Pediatric Allergy and Immunology : Official Publication of the European Society of Pediatric Allergy and Immunology·Norio KawamotoNaomi Kondo
Nov 17, 2009·American Journal of Reproductive Immunology : AJRI·Ageliki PantaziMarighoula Varla-Leftherioti
Feb 14, 2006·American Journal of Transplantation : Official Journal of the American Society of Transplantation and the American Society of Transplant Surgeons·G WestallA Brooks
Mar 5, 2002·Clinical and Diagnostic Laboratory Immunology·Nancy J O'Neil-Andersen, David A Lawrence
Dec 22, 1999·The Journal of Immunology : Official Journal of the American Association of Immunologists·R CambroneroJ Farrant
Oct 4, 2006·The Journal of Immunology : Official Journal of the American Association of Immunologists·Michael WaltherEleanor M Riley
Nov 22, 2018·Veterinary Immunology and Immunopathology·Guilherme De Caro MartinsDenise da Silveira Lemos

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