Determination of frequencies of Th17 cells in human peripheral blood by flow cytometry
10.3969/j.issn.1673-4130.2014.15.044
- VernacularTitle:流式细胞术检测人外周血Th17细胞频率
- Author:
Ping XIAO
;
Zuchang WU
;
Wei LIN
- Publication Type:Journal Article
- Keywords:
flow cytometry;
Th17cells;
activation,in vitro
- From:
International Journal of Laboratory Medicine
2014;(15):2069-2070,2072
- CountryChina
- Language:Chinese
-
Abstract:
Objective To investigate the effect of different stimulating culture conditions and detecting methods on the frequency of Th17 cells and to set up and optimize the experimental method for detecting the frequencies of human Th 17 cells in peripheral blood by flow cytometry (FCM ) .Methods The peripheral blood in the healthy people was performed the stimulating culture by phorbol myristate acetate (PMA) at different final concentrations(50 ,100 ,150 ,200 ,250 ,300 ng/mL) and ionomycin 1 μg/mL .The frequencies of Th17 cells were assessed by FCM after different stimulating time points (4 ,6 ,8 ,10 ,12 h) .The mean fluorescence in-tensity(MFI) and percentage of the ratio of CD4 positive cells on the cellular surface and inside cell membrane were measured be-fore and after PMA stimulation .Results The frequency of Th17 cells was highest with the good stimulation effect under the condi-tion of PMA 150 ng/mL and ionomycin1μg/mL stimulation for 6 h (P<0 .05) .MFI of CD4 inside cellular membrane after stimula-tion was lower than that in the cells with stimulation after labeling membrane surface CD 4(P<0 .05) ,and the Th17 cell frequencies and the ratio of CD4 positive cells were higher than those in the latter ,but the differences were not statistically significant (P>0 .05) .Conclusion After stimulating whole blood with the final concentration of PMA 150ng/mL and ionomycin1μg/mL for 6 h , measuring intracytoplasm CD4 and IL-17 levels is the simple and effective method for detecting the frequency of human Th 17 cells .
