Expression and kinetics of cytokines determined by intracellular staining using flow cytometry
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Expression and kinetics of cytokines determined by intracellular staining using flow cytometry. / Mascher, B; Schlenke, P; Seyfarth, M.
In: J IMMUNOL METHODS, Vol. 223, No. 1, 01.02.1999, p. 115-21.Research output: SCORING: Contribution to journal › Short publication › Research › peer-review
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TY - JOUR
T1 - Expression and kinetics of cytokines determined by intracellular staining using flow cytometry
AU - Mascher, B
AU - Schlenke, P
AU - Seyfarth, M
PY - 1999/2/1
Y1 - 1999/2/1
N2 - Cytokine production by human lymphocytes was assessed by a flow cytometric procedure involving staining of intracellular cytokines by the paraformaldehyde-saponin procedure. The production of interleukin 2 (IL2), interferon gamma (IFNgamma) and tumor necrosis factor alpha (TNFalpha) was determined in T-helper (Th) and cytotoxic T-cells (CTL) as well as in naive and memory cells after stimulation with phorbol-12-myristate-13-acetate (PMA) and ionomycin under the influence of monensin. Kinetic studies on IL2, IFNgamma and TNFalpha in lymphocyte subpopulations showed that TNFalpha was the first cytokine produced by T-cells. Production of this cytokine peaked at 2 h and declined rapidly thereafter. The peak of IL2 and IFNgamma production was at 8 h, and production of IL2 exceeded that of IFNgamma in T-cells at all times. IL2 production declined markedly after 8 h, while IFNgamma remained relatively stable for 24 h. IL2 and TNFalpha were mainly produced by Th cells while CTL primarily expressed IFNgamma. At all times a higher percentage of memory cells stained cytokine positive compared to naive cells and production of cytokines increased more rapidly in the memory cells. Naive cells produced primarily IL2, while memory cells expressed all the studied cytokines in substantial amounts. Kinetic studies between 1 and 24 h showed that 5 h was the optimal time point for evaluating the cytokines studied; hence normal values obtained from 50 healthy blood donors were evaluated after 5 h continuous PMA and ionomycin stimulation.
AB - Cytokine production by human lymphocytes was assessed by a flow cytometric procedure involving staining of intracellular cytokines by the paraformaldehyde-saponin procedure. The production of interleukin 2 (IL2), interferon gamma (IFNgamma) and tumor necrosis factor alpha (TNFalpha) was determined in T-helper (Th) and cytotoxic T-cells (CTL) as well as in naive and memory cells after stimulation with phorbol-12-myristate-13-acetate (PMA) and ionomycin under the influence of monensin. Kinetic studies on IL2, IFNgamma and TNFalpha in lymphocyte subpopulations showed that TNFalpha was the first cytokine produced by T-cells. Production of this cytokine peaked at 2 h and declined rapidly thereafter. The peak of IL2 and IFNgamma production was at 8 h, and production of IL2 exceeded that of IFNgamma in T-cells at all times. IL2 production declined markedly after 8 h, while IFNgamma remained relatively stable for 24 h. IL2 and TNFalpha were mainly produced by Th cells while CTL primarily expressed IFNgamma. At all times a higher percentage of memory cells stained cytokine positive compared to naive cells and production of cytokines increased more rapidly in the memory cells. Naive cells produced primarily IL2, while memory cells expressed all the studied cytokines in substantial amounts. Kinetic studies between 1 and 24 h showed that 5 h was the optimal time point for evaluating the cytokines studied; hence normal values obtained from 50 healthy blood donors were evaluated after 5 h continuous PMA and ionomycin stimulation.
KW - Cytokines/biosynthesis
KW - Flow Cytometry/methods
KW - Humans
KW - Immunophenotyping
KW - Interferon-gamma/biosynthesis
KW - Interleukin-2/biosynthesis
KW - Intracellular Fluid/chemistry
KW - Ionomycin/pharmacology
KW - Kinetics
KW - Leukocytes, Mononuclear/chemistry
KW - Staining and Labeling
KW - T-Lymphocyte Subsets/chemistry
KW - Tetradecanoylphorbol Acetate/pharmacology
KW - Time Factors
KW - Tumor Necrosis Factor-alpha/biosynthesis
U2 - 10.1016/s0022-1759(98)00200-2
DO - 10.1016/s0022-1759(98)00200-2
M3 - Short publication
C2 - 10037239
VL - 223
SP - 115
EP - 121
JO - J IMMUNOL METHODS
JF - J IMMUNOL METHODS
SN - 0022-1759
IS - 1
ER -