In our lab, we have know-how about performing MPLC assays, such as using it in the establishment of immunomonitoring vaccination trials with RNA transfected DC by using overlapping 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 expertise in lipofection of tumor-antigen-encoding RNA into DC. The method was compared to RNA electroporation into DC.

We have experience in multicolour flow cytometry. Besides ELISPOT, tetramer based 8-colour flow cytometry has become our routine monitoring assay, allowing extended phenotypic and functional characterization of T cell subpopulations. Of special interest has become the identification of polyfunctional T cells (capable to simultaneously exert several functions, i.e. production of various cytokines and killing activity) since these have been shown to elicit more effective immune responses in HIV vaccination trials.

There is a MACS separator (Miltenyi Biotech) at the disposal of our lab. It is used, for example, to purify CD8 T cells.

A Luminex flow cytometer is available to our lab. It is used, among other things, to quantify the TNFalpha and IFNgamma secretion in a study on antigen recognition.

There is a Luminex flow cytometer available in the lab. It is used, among other things, for assaying of cytokines in B-CLL patients treated with Apo-DC +/- GM-CSF.

We have developed immunological techniques for monitoring of tumour antigen specific CD4+ T cell responses. We set an in vitro system able to detect fona fide in vivo primed CD4+ T cells. Purified CD4+ T cells are first stimulated in vitro once in the presence peptides, previously identified as containing naturally processed epitopes on tumour-associated antigens. After two weeks, proliferation and cytokine secretion (IFN-?, IL-5, IL-4, IL-2, TNF-?, IL-10, TGF-?, IL-17, granzyme and perforin) are tested. To estimate the frequency of antigen specific CD4+ T cell we us...

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. To this aim, iterative calibration algorithms were used to achieve a final, absolute mass accuracy of better than 10 parts per million (p.p.m.) for all peptide ions.

The integral plasma membrane proteome of D1 cells stimulated for 24 hours with LPS has been analyzed with this LC-MS/MS analyser and compared to the results obtained from immature D1 cells.

We have know-how about LDA assays; for instance, we use LDA assays to monitor vaccine induced T cell responses.

In our labs we have experience in performing immunohistochemistry, e.g., for studying the cellular organization of the marrow of long bones.

There is a GMP facility for conduction of phase III clinical trials available. The structural up-grade of this facility has limited the number of patients recruited.

The FACS analyzer avaiable to the lab is used, among other things, to assess protein expression of tumor antigens or soluble proteins.

The gene gun system available to the laboratory was used, e.g., for micro-particle bombardment of murine skin.

The Genopolis microarray database serves as an easy interface for the data generated by the Affymetrix GeneChip platform. DC transcriptomic data produced in the past 4 years has made been an accessible resource for data sharing and data integration accessible to all the DC community. The experiments on mice and rat are currently being annotated and conserved in the Genopolis database. Direct access to the primary data held in the Biopolo database is responsibility of the BIOPOLO Project Management System (see http://www.biopolo.it/).

Our laboratory has expertise in applying a fluorescence microscopy method to analyse proximal signalling events induced by cross-linking C-type lectins.

Experience has been gained in identifying DC presenting a specific peptide via a sensitive immunofluorescent staining technique.

The FAX analyser available in the laboratory is used, e.g., to measure transfection efficiency.

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...