(A new protocol will probably be started in the near future.)

BruCells has been actively involved in the discussion of the EMEA guideline on Cell Based Medicinal Products (CBMP). Catherine De Greef was present at the DC-THERA Cluster 4 Meeting in Bamberg (July 2007) where among other topics these guidelines were discussed and where the basis for a written statement has been worked out. During the finalisation of the statement, BruCells has mainly put forward the notion that the guidelines are not suitable to CBMP for the authorization of investigational trials, but are applicable in the procedure of obtaining market approval.

We have developed a technology to obtain from melanoma metastases melanin-free total tumor RNA, suitable for in vitro amplification and DC transfection.

The optimization for the mass spectrometry analysis methods to be used in the generation of a initial proteomic map of mouse DCs (WP2) and stimuli-related phosphorylation cascades (WP5) was carried out by Lyris M. F. de Godoy (Post-doc) and Jesper V. Olsen (PhD student). Recently, our lab has been developing new techniques in quantitative proteomics and has also acquired new instrumentation to assess these types of questions in depth. This, of course, involves a lot of optimization and, consequently, large amounts of sample. However, to obtain high numbers of dend...

To generate a proteomic map of DCs (WP2) which includes quantitative information that can be used further to study signalling cascades (WP5) it is also necessary to optimize the SILAC labelling of DCs. This work was performed by Christian A. Luber (PhD student). First, we have set up a system for SILAC labelling of bone marrow derived murine dendritic cells (BM-DC). The principal method for generating BM-DC with GM-CSF was adapted from Lutz et al. (J Immunol Methods 1999, 223: 77-92). After some modifications of the protocol the FACS analysis of DC, which were gen...

Pathway analysis performed with Eu.Gene (Cavalieri et al., 2005), following the algorithm developed by Beltrame et al. (2009).

A protocol for obtaining highly pure integral membrane proteins has been set up in the last two months in the lab of Paola Castagnoli in collaboration with the lab of Juri Rappsilber at IFOM.

Protocols were developed to refold in vitro CD1 molecules, which were used for the generation of CD1 tetramers. The ability to generate CD1d tetramers has provided us with the opportunity of comparing a broad panel of CD1d binding compounds for their ability to stimulate iNKT cells.

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.

Protocol for the documentation and the analysis of the interaction between RIG-I, a viral sensing protein, and NS1, an inhibitor of interferon production that is encoded by influenza A virus. This protocol has been made available.

Array construction: The yeast oligonucleotide array was constructed using the S. cerevisiae Genome Oligo Set ™ (Operon Technologies, CA, USA) composed of 6240 optimized oligonucleotides (70mers) each representing one yeast gene. Probe preparation and hybridization: We used the indirect labeling method. Briefly, the reactive amine derivative of dUTP, 5-(3- aminoallyl)-2?-deoxyuridine 5?-triphosphate (Sigma) was incorporated into cDNA using the Superscript II reverse transcriptase (Invitrogen) and oligo dT (Invitrogen) and random examers (Roche). After synthesi...