The clinical application of flow cytometry for detecting intracellular IL-2 in γδΤ cells

doi:10.3877/cma.j.issn.2095-655X.2015.04.018

Abstract

Abstract:

Objective

To establish a method for detecting intracellular IL-2 in human peripheral blood γδΤ cells.

Methods

Healthy human peripheral blood mononuclear cells (PBMCs) were isolated, and then stimulated with low molecular peptide antigen of mycobacterium tuberculosis (Mtb-Ag). A high proportion of γδΤ cells were obtained.PBMCs and γδΤ cells were excited with phorbol myristate acetate (PMA) and ionomycin(IM). Protein transport inhibitor (Brefeldin A, BFA) was used to block the secretion of cytokines and saponin was taken for membrane permeabilization.Flow cytometry was applied to detect the expression of CD69 in CD³⁺ T cells to explore the appropriate time of membrane permeabilization.Then flow cytometry was implemented to detect the expression of intracellular IL-2 in γδΤ cells.Analysis of Variance (ANOVA) and Least Significant Difference(LSD) test were conducted to determine the statistical difference.

Results

CD³⁺ T cells with 15 minutes of membrane permeabilization had the highest expression of CD69 (87.82±2.28)% compared with those with 5 minutes and 25 minutes (F=112.81, P<0.05). γδΤ cells with the stimulation of PMA, IM and blocker of BFA (γδΤ+ PMA+ IM+ BFA) had the highest expression of intracellular IL-2 (50.65±6.25)% compared with γδΤ group, γδΤ+ PMA+ IM group and PBMC+ PMA+ IM+ BFA group(F=321.07, P<0.05).

Conclusion

Flow cytometry is an appropriate method for the detection of intracellular IL-2 in γδΤ cells, and it may be applicable to detecting other cytokines in γδΤ cells.

Key words: γδΤ cells, Interleukin-2, Phorbol myristate acetate, Ionomycin, Antigen of mycobacterium tuberculosis

Keqiang Wang, Yanqiang Hou, Zhangshen Ran, Xiangqi Li, Yanchao Duan. The clinical application of flow cytometry for detecting intracellular IL-2 in γδΤ cells[J]. Chinese Journal of Diagnostics(Electronic Edition), 2015, 03(04): 292-295.

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Figures/Tables 3

References 11

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破膜时间(min)	样本数	CD3^＋T细胞阳性率	CD3^＋T细胞内CD69表达
5	5	68.25±2.42	66.86±1.99
15	5	66.16±2.13^a	87.82±2.28^d
25	5	58.37±1.89^bc	81.73±2.51^ef
F值	?	29.12	112.81
P值	?	<0.05	<0.05

破膜时间(min)	样本数	CD3^＋T细胞阳性率	CD3^＋T细胞内CD69表达
5	5	68.25±2.42	66.86±1.99
15	5	66.16±2.13^a	87.82±2.28^d
25	5	58.37±1.89^bc	81.73±2.51^ef
F值	?	29.12	112.81
P值	?	<0.05	<0.05

组别	样本数	细胞表面	细胞内
PBMC组	5	0.66±0.08	0.50±0.06
PBMC＋PMA＋IM组	5	63.73±7.25^a	2.79±0.31^d
PBMC＋PMA＋IM＋BFA组	5	4.56±4.06^bc	57.12±0.62^ef
F值	?	242.78	31826.16
P值	?	<0.01	<0.01

组别	样本数	细胞表面	细胞内
PBMC组	5	0.66±0.08	0.50±0.06
PBMC＋PMA＋IM组	5	63.73±7.25^a	2.79±0.31^d
PBMC＋PMA＋IM＋BFA组	5	4.56±4.06^bc	57.12±0.62^ef
F值	?	242.78	31826.16
P值	?	<0.01	<0.01

组别	样本数	IL-2表达
γδΤ细胞组	5	0.55±0.07
γδΤ细胞＋PMA＋IM组	5	0.91±0.12^a
γδΤ细胞＋PMA＋IM＋BFA组	5	50.65±6.25^bc
PBMC＋PMA＋IM＋BFA组	5	0.21±0.03^def
F值	?	321.07
P值	?	<0.01

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