We have knowhow on ELISPOT analysis of long peptide vaccines.

Our lab has expertise with Elispot assays. We evaluated the capacity of autologous DC and HEK293 cells transfected with relevant HLA alleles to function as T cell targets in Elispot assays upon transfection with tumor antigen encoding plasmids.

We have experience with the exponentially modified Protein Abundance Index (emPAI) method, a label free spectral counting procedure for determining relative protein amounts of LC-MS/MS data. 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. The criteria applied resulted in a list of 2794 unique proteins that were identifie...

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

We have experience on performing immunofluorescence assays. As an example, an assay on ordered sequential sections was used to analyze the infiltration and destruction of solid tumors by CTLs.

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

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

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...

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

We have know-how about performing LDA assays. We have, for instance, monitored vaccine induced T cell responses for the two clinical trials. We found CD8+ and CD4+ T cell responses against the immunological tracers KLH and TK, and against the NTPs and MAGE-3, respectively.

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

The laboratory has expertise in the use of MACS anti-CD14 microbeads by Miltenyi Biotec, Bergisch-Gladbach, Germany. They were used for isolation of monocytes from PBMCs.

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.

In our laboratory we carry out multiplex assays. For instance, multiplex analysis was used to study the response of human MoDC to yeast, spheroplasts, pseudohyphae and spores.

Using this peptide library technique and ELISPOT analysis patients´ PBMC are pre-screened for CD4- and CD8- T cell responses and individual sets of 4 to 8 peptides chosen for further analysis.

In our work, we have experience in applying peptide-tetramer assays. Primary and secondary CTL response against the C8 T cell epitope SIINFEKL was monitored in a study for maturing myeloid DCs and plasmacytoid DCs.

Using MAGE-A3 as a model antigen we established a functional read-out system by transfection of CD4 T cells with RNA encoding T cell receptors recognizing MHC DP4 restricted MAGE-A3 epitopes.

Real time and immunohistochemistry analysis was performed in a study to evaluate whether iNOS can serve as a prognostic factor in stage III melanoma survival. The analysis showed that both iNOS and COX-2 alone significantly predicted OS. The BRAF/NRAS mutation status did not significantly differ between patient groups, although iNOS significantly correlated with BRAF mutation frequency. Furthermore, iNOS and COX-2 were significantly linked to a number of metastatic lymph nodes. However, iNOS maintained its statistical significance together with a number of lymph node...

In our lab we have experience in performing real time PCR analyses.

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...