Our lab has expertise in lipofection of tumor-antigen-encoding RNA into DC. The method was compared to RNA electroporation into DC.

Argos has made extensive progress in automating a closed, GMP- compliant system for DC manufacturing. This system involves a computer-controlled workstation that links a GAMBRO Elutra device (to harvest monocytes from pheresis products) to closed-system cell culture process. All media changes and washes are automated and limited operator intervention is required throughout the entire process. A second computer-controlled work station harvests the matured DC, electroporates them with RNA, and aliquots, vials, and freezes individual vaccine doses. Argos has consulted wi...

A Gambro BCT Elutra Cell Separation System is available to us. We have experience, e.g., in using it for monocyte enrichment.

We have used the electron microscope that is available to our lab, e.g., to document the production and secretion of tubulovesicular structures by cells overexpressing VSV-G glycoprotein and for documentation of production and secretion of tubulovesicular structures.

Led by Figdor (P2), this platform was established between four centres that respectively provide expertise and facilities for a variety of high resolution imaging techniques and MRI of humans in Nijmegen NL (P2, Figdor); MRI of mice in Erlangen DE (P6, Lutz); two photon microscopy in Paris FR (P13, Amigorena); and correlation spectroscopy in Marseilles FR (P22, Pierre). The platform organised several high quality courses and workshops in 2006-7 which continued in 2008.

A confocal microscope is available in our laboratory.

In our institute, we have a 9-colour FACS cell sorter our disposal. It is used, e.g., for the analysis of various DC populations.

Genopolis hosts a microarray platform that uses the Affymetrix GeneChip® microarray technology for genome and transcriptome analyses. In 2008, this platform was enhanced by the incorporation of a new institute [the Wellcome Trust Centre for Human Genetics] to provide ‘Illumina’ microarray profiling. The following services are currently available: Whole genome genotyping applications, which include: 1. family-based association studies 2. cancer genetics 3. population genetics 4. chromosomal copy number analysis Whole genome gene expression profiling application...

Our laboratory has an electroporator at its disposal. It is used for instance for RNA electroporation into DC.

We have experience in querying DAVID, the Database for Annotation, Visualization and Integrated Discovery (http://david.abcc.ncifcrf.gov/) . For instance, we used DAVID for functional annotation of upregulated and downregulated genes by gene ontology classification and pathway analysis by several bioinformatics approaches. Preliminary results obtained from querying DAVID and Ontologizer (http://www.charite.de/ch/medgen/ontologizer/) show statistically significant enrichment for immunological-relevant signaling pathways and immunology-related GO terms of the upregula...

We have access to and experience with using a linear ion trap / Fourier Transform (LTQ-FT) hybrid mass spectrometer. For example, we use it to analyse purified tryptic peptides.

A comprehensive protein inventory of clinical grade immature and cytokine cocktail matured (Il-6, IL-1 beta, TNF alpha, PGE2; 48 hours) monocyte derived human dendritic cells (DC) from a healthy donor has been established by using high accuracy, high sensitivity protein identification technology. We have identified 2794 proteins in DCs by liquid chromatography tandem mass spectrometry.

Our lab has experience in carrying out ELISPOT analyses. For instance, an IFNgamma ELISPOT assay with PBMC loaded with peptide pools (each consisting of 10 15-mers, which overlap with 11 aminoacids) was done for a study on antigen recognition.

We have developed, at the Curie Institute, a unique methodology dedicated to the identification of phagosome proteins in dendritic cells, under various experimental conditions. This methodoly incorporates a unique sample preparation procedure based on 96 well plate complex sample trypsinization, a nano-LC-based peptide separation coupled to MSMS analysis sequencing. This procedure led us to the identification of 600 phagosomal proteins from murine DC phagosomes. An integrated software solution has been developed to analyse these results and to perform comparative analy...

This puromycin-based technology was developed in our laboratory to monitor translation by FACs in individual or cell populations. It is a non-invasive method to monitor protein synthesis and cellular activation in single cells or heterologous populations.

The MaxQuant software is designed to make it possible to compare cell subsets quantitatively based on the extracted ion current (XIC) and without any need of stable isotope labels.

Mass spectrometry (MS)-based proteomics has become a powerful technology to map the protein composition of organelles, cell types and tissues. In our department, a large-scale effort to map these proteomes is complemented by the Max-Planck Unified (MAPU) proteome database. MAPU contains several body fluid proteomes; including plasma, urine, and cerebrospinal fluid. Cell lines have been mapped to a depth of several thousand proteins and the red blood cell proteome has also been analyzed in depth. The liver proteome is represented with 3200 proteins. By employing hi...

For quantitative proteomic studies, our group recently described the SILAC (Stable Isotope Labelling by Amino Acids in Cell Culture) approach. In this method, independent populations of cells are grown in medium containing different non-radioactive isotope forms of essential amino acids (e.g. arginine and lysine). Because the cells cannot synthesize these amino acids, after some generations all proteins are labelled and therefore can be distinguished and quantified by the mass spectrometer. To generate a proteomic map of DCs which includes quantitative information that...

We have a FACS Aria cell sorter at our disposition from BD Biosciences (URL: http://www.bdbiosciences.com/features/products/display_product.php?keyID=53). We used this FACS Aria among other things for fluorescence activated cell sorting for proteomic investigations. Using Fluorescence activated cell sorting allowed us to obtain highly pure fractions of CD8+, CD4+ and DN DC subsets. However, because DC are a very rare population, this method is very material- and cost-intensive. Furthermore, it is not surprising that the obtained cell numbers of each subset after isola...

There is a confocal microscope at our disposal for confocal imaging.