The surface protein composition of extracellular vesicles (EVs) is related to the originating cell and may play a role in vesicle function. Knowledge of the protein content of individual EVs is still limited because of the technical challenges to analyse small vesicles. Here, we introduce a novel multiplex bead-based platform to investigate up to 39 different surface markers in one sample. The combination of capture antibody beads with fluorescently labelled detection antibodies allows the analysis of EVs that carry surface markers recognized by both antibodies. This new method enables an easy screening of surface markers on populations of EVs. By combining different capture and detection antibodies, additional information on relative expression levels and potential vesicle subpopulations is gained. We also established a protocol to visualize individual EVs by stimulated emission depletion (STED) microscopy. Thereby, markers on single EVs can be detected by fluorophore-conjugated antibodies. We used the multiplex platform and STED microscopy to show for the first time that NK cell–derived EVs and platelet-derived EVs are devoid of CD9 or CD81, respectively, and that EVs isolated from activated B cells comprise different EV subpopulations. We speculate that, according to our STED data, tetraspanins might not be homogenously distributed but may mostly appear as clusters on EV subpopulations. Finally, we demonstrate that EV mixtures can be separated by magnetic beads and analysed subsequently with the multiplex platform. Both the multiplex bead-based platform and STED microscopy revealed subpopulations of EVs that have been indistinguishable by most analysis tools used so far. We expect that an in-depth view on EV heterogeneity will contribute to our understanding of different EVs and functions.
Rhodobacter capsulatus NtrB/NtrC two-component regulatory system controls expression of genes involved in nitrogen metabolism including urease and nitrogen fixation genes. The ntrY-ntrX genes, which are located immediately downstream of the nifR3-ntrB-ntrC operon, code for a two-component system of unknown function. Transcription of ntrY starts within the ntrC-ntrY intergenic region as shown by primer extension analysis, but maximal transcription requires, in addition, the promoter of the nifR3-ntrB-ntrC operon. While ntrB and ntrY single mutant strains were able to grow with either urea or N2 as sole nitrogen source, a ntrB/ntrY double mutant (like a ntrC-deficient strain) was no longer able to use urea or N2. These findings suggest that the histidine kinases NtrB and NtrY can substitute for each other as phosphodonors towards the response regulator NtrC.
The differentiation of CD4+ or CD8+ T cells following priming of naive cells is central in the establishment of the immune response against pathogens or tumors. However, our understanding of this complex process and the significance of the multiple subsets of differentiation remains controversial. Gene expression profiling has opened new directions of investigation in immunobiology. Nonetheless, the need for substantial amount of biological material often limits its application range. In this study, we have developed procedures to perform microarray analysis on amplified cDNA from low numbers of cells, including primary T lymphocytes, and applied this technology to the study of CD4 and CD8 lineage differentiation. Gene expression profiling was performed on samples of 1000 cells from 10 different subpopulations, defining the major stages of post-thymic CD4+ or CD8+ T cell differentiation. Surprisingly, our data revealed that while CD4+ and CD8+ T cell gene expression programs diverge at early stages of differentiation, they become increasingly similar as cells reach a late differentiation stage. This suggests that functional heterogeneity between Ag experienced CD4+ and CD8+ T cells is more likely to be located early during post-thymic differentiation, and that late stages of differentiation may represent a common end in the development of T-lymphocytes.
Solid tumors consist of heterogeneous cell types including a subpopulation of stem like cells. Starting from bulk tumor mass, the analysis of these cancer stem cells (CSCs) is hampered by their rare frequency and their characteristics remain hidden. To address this issue, we have developed a method for the semi-automated dissociation of human tumor tissue yielding a viable single cell suspension. Subsequently, a magnetic cell sorting (MACS) strategy was established allowing for the isolation of CD24-/CD44+ breast CSCs. Using these methods, we isolated breast CSCs from an invasive ductal carcinoma at a purity of 94%. Due to the limited starting material, a few hundred CSCs as well as bulk tumor cells were used for global PCR based RNA amplification. The amplified cDNA was sheared, size selected, and processed for high throughput gene expression analysis on the Illumina Genome Analyzer system. Samples were prepared in triplicate and over 10 million individual purity-filtered reads were analyzed per sample. In addition, Agilent whole genome microarrays were used for validation of expression differences. Analysis of the sequence tags allowed us to identify novel putative markers overexpressed in breast CSCs. GeneOntology analysis was used to determine functional groups of genes which are differentially regulated between CSCs and bulk tumor cells. We found a strong overrepresentation of genes connected to TGF-beta as well as Wnt/GSK3 signaling supporting the correlation of the CSC phenotype with epithelial-mesenchymal-transition (EMT). Other differentially expressed genes are known to be involved in pluripotency, cell migration, and apoptosis further indicating a stem cell-like fate.Our proof of principle experiment demonstrates the advantage of isolating rare cell populations for subsequent molecular analysis by high throughput sequencing. The analysis of purified CSCs revealed new insights on the regulation, maintenance, and biology that would not have been observed when assessing only the bulk tumor mass. Analyzing the genetic set up of defined tumor subpopulations by Next Generation Sequencing can improve our understanding of cancer and could pioneer targeted diagnostic and therapeutic approaches. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3329. doi:10.1158/1538-7445.AM2011-3329
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