[Cytokine expression of CD4+ and CD8+ T cells induced by different stimuli under different culture conditions].
OBJECTIVE
To investigate the effect of different stimuli and different culture conditions on the cytokine expression of CD4+ and CD8+ T cells.


METHODS
PBMCs were isolated from normal human peripheral blood and cultured with three kinds of stimuli (PHA, anti-CD3 and anti-CD28 mAbs, PMA and ionomycin) under four different culture conditions (room temperature, 37 degrees C water bath, 37 degrees C incubator, 37 degrees C and 50 mL/L CO2 incubator) for 4.5 ~ 5 h and intracellular cytokines (IL-2, IFN-gamma and TNF-alpha) in T cells were assessed by flow cytometry (FCM).


RESULTS
Cytokine expression of CD4+ and CD8+ T cells varied when treated with different stimuli under different conditions. PMA had the strongest stimulative effect on cytokine expression of PBMC, while the effect of anti-CD3 mAb was weaker, and that of PHA was the weakest. Different culture conditions did not greatly change the cytokine expression profile of T cells (P>0.05) except that there were very few cytokine producing cells when PBMCs were cultured at room temperature.


CONCLUSIONS
Anti-CD3 and anti-CD28 mAbs, PMA and ionomycin are recommended for the stimulation of PBMCs and detection of intracellular cytokine using FCM. Culture temperature but not the concentration of CO2 is the most important factor during the short term T cell stimulation.
Zhou
Mao-hua
MH
Department of Immunology, Medical School, Sun Yat-sen University, Guangzhou 510080, China. maohuazhou@126.com
Wu
Chang-you
CY
D000328:Adult; D000906:Antibodies / Q000276:immunology / Q000494:pharmacology; D018106:CD28 Antigens / Q000276:immunology; D017252:CD3 Complex / Q000276:immunology; D015496:CD4-Positive T-Lymphocytes / Q000187:drug effects / Q000378:metabolism*; D018414:CD8-Positive T-Lymphocytes / Q000187:drug effects / Q000378:metabolism*; D005260:Female; D005434:Flow Cytometry; D005786:Gene Expression Regulation / Q000187:drug effects; D006801:Humans; D007371:Interferon-gamma / Q000378:metabolism; D007376:Interleukin-2 / Q000378:metabolism; D015759:Ionomycin / Q000494:pharmacology; D007476:Ionophores / Q000494:pharmacology; D008297:Male; D013696:Temperature; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism
D000906:Antibodies; D018106:CD28 Antigens; D017252:CD3 Complex; D007376:Interleukin-2; D007476:Ionophores; D014409:Tumor Necrosis Factor-alpha; D015759:Ionomycin; D007371:Interferon-gamma