Dataset features


Application: Gene expression microarray analysis
Number of samples: 11
Release date: Sep 6 2011
Last update date: Dec 3 2015
Access: Public
Diseases: Hypophosphatemia, Familial, Pneumonia of Calves, Enzootic
Chemicals: Fucose, Phosphates
Dataset link Glyco-gene expression in naive and activated murine regulatory T-cells vs. conventional T-cells

Experimental Protocol

Preliminary studies are underway to ascertain the minimum medium required to sustain general T cell survival, whilst maintaining ‘clean’ glycomic profiles. The effect of glycoprotein withdrawal on CD25+ and CD25- CD4+ T cells in culture was investigated by selecting the cells in FCS-free medium and incubating them for six hours with either FCS-containing medium, or PBS supplemented with FCS, fetuin or fetuin-derived glycans. Early apoptosis was measured using Annexin-V (BD Biosciences, Heidelberg, Germany) and late apoptosis using 7-aminoactinomycin D (7-AAD) (BD Biosciences), in parallel with a complementary apoptosis detection method – measurement of the mitochondrial membrane potential using the BD™ Mitoscreen Kit (BD Biosciences). This apoptosis staining study demonstrated that there was a remarkably higher percentage of death in both CD25+ and CD25- CD4+ T cells cultured in PBS versus cells cultured in RPMI-1640 or medium containing FCS. Both CD25+ and CD25- CD4+ T cells were highly susceptible to apoptosis in glycoprotein-free medium, demonstrating their exquisite sensitivity to the lack of glycoprotein survival factors. Continuing studies are addressing the minimum medium required to sustain cells undergoing polyclonal activation in vitro and the impact of activation on the N-glycan profiles of Tregs versus conventional T cells. To complement this work, we used Core E resources to investigate glycosyltransferase gene expression in freshly isolated versus activated C57BL/6 CD25+ and CD25- CD4+ T cells. Three populations of murine CD4+ T cells were isolated from the spleens of C57BL/6 mice by flow-assisted cell sorting: CD25+CD45RBlow, CD25-CD45RBhigh and CD25-CD45RBlow. An RNeasy mini kit (QIAGEN, Manchester, UK) was used for total RNA extraction, following the manufacturer’s protocol. A microarray assay was performed using the Glyco-gene Chip v4, containing 1,127 probe sets targeting glycosylation-related transcripts including genes encoding proteins that are either heavily glycosylated or involved in glycosylation, as well as 119 probe sets targeting reference (‘housekeeping’) transcripts. Robust Multichip Average (RMA) software was used to convert intensity to expression values.










Steven Head