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Peripheral Blood TIM-3 Positive NK and CD8+ T Cells throughout Pregnancy: TIM-3/Galectin-9 Interaction and Its Possible Role during Pregnancy
〖 2014-03-20 | 点击 1005 〗
Abstract

Problem

The T-cell immunoglobulin and mucin domain (TIM) family is a relatively newly described group of molecules with a conserved structure and important immunological functions. Identification of Galectin-9 as a ligand for TIM-3 has established the Galectin-9/TIM-3 pathway as an important negative regulator of Th1 immunity and tolerance induction. Data about the TIM-3/Gal-9 pathway in the pathogenesis of human diseases is emerging, but their possible role during human pregnancy is not precisely known. The aim of our study was to investigate the number, phenotype and functional activity of TIM-3+ peripheral blood mononuclear cells during healthy human pregnancy. . . . .

Galectin-9 ELISA

Serum samples were collected from healthy non-pregnant individuals and from the 1st, the 2nd and the 3rd trimester group of gestation. 10ml venous blood was taken to disposable sterile test tube and centrifuged at 2000 rpm for 10 minutes. Serum samples then transferred in 1ml aliquots into cryovials and stored frozen at –80°C until analysis.

Serum Gal-9 levels were measured by ELISA in 26 non-pregnant women and in 10 healthy women in each trimester of pregnancy.

The serum Gal-9 concentration was determined by a quantitative sandwich enzyme immunoassay according to the manufacturer’s protocol (BLUEGENE GAL9 ELISA kit, AMS Biotechnology, UK). Briefly, 50 μl/well of standard and human serum samples obtained from healthy pregnant woman and normal controls were added to 96 well microplate pre-coated with monoclonal antibody specific for Gal-9 protein. Then 100 μl of enzyme conjugate (horseradish peroxidase-conjugated polyclonal anti-Gal-9) was added to each well, mixed thoroughly and incubated for 1 hour at 37°C. Then the wells of the microtiter plate were washed 5 times with 200 μl of Wash buffer. For color development 50 μl of Substrate A and 50 μl of Substrate B were added to each well, and incubated for 10–15 minutes at 37°C in the dark. Finally 50 μl/well of Stop solution was added to the wells and mixed with gentle tapping to terminate the reaction. The optical density was measured at 450nm with FluoSTAR Optima (BMG Labtech, Germany) microplate spectrophotometer and the Gal-9 protein concentration was determined with Optima 2.10 R2 built-in data calculator software.


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