Methods
Adaptive Immune System
New Protocols added! (09.06.11)
- Isolation of PBMCs
In this protocol we explain our methods to isolate, freeze and thaw peripheral blood mononuclear cells for functional assays. Protocol: Isolsation using Ficoll: Download pdf-Format, Protocol: PBMC Isolation, Freezing and Thawing Download pdf-Format - MHC Multimer Analysis
The multimer analysis allows the identification of antigen-specific T cells independent of their function but calls for the knowledge of the MHC. Protocol for fixed cells: Download pdf-format, for unfixed cells: Download pdf-format - Intracellular Cytokine Staining
With the help of intracellular cytokine staining (ICS) it is possible to identify functional antigen-specific T cells. In combination with multimer analysis and staining of cell surface molecules by means of multicolor flow cytometry this method allows for complex quantitative evaluation of vaccine-induced immunogenicity. Download pdf-Format - Flow Cytometry School
We help you with the basics of multicolor flow cytometry analysis. - Cytokine Capture Assay
This cytokine capture assay provides identification and enrichment of tumor- or pathogen reactive T cells (CD4+ and CD8+) by their effector function without prior knowledge of the specific antigen and the MHC profile. - ELISPOT Assay
The ELISPOT assay is one of the most sensitive assays to quantify antigen-specific T cells at the single cell level via secretion of effector molecules such as cytokines, granzyme B and perforin. In general, it does not recquire in vitro stimulation or exogenous cytokines, it works with fresh and frozen cells and is often the first choice in vaccine evaluation and mapping of T cell epitopes. Download pdf-format - Multiplex Bead Array
These assays provide a quantitative analysis of a variety of cytokines, chemokines or phosphoproteins simultaneously in small-volume samples of patient serum, plasma or culture supernatant following in vitro stimulation (e.g. BioPlex Luminex technology or BD CBA FlexSet system using a flow cytometer) - ELISA
The ELISA (enzyme-linked immunosorbent assay) is useful for measuring antibody responses in the plasma or serum of the patients. Most of the cytokines can be better quantified by the other technologies. - Immunohistology
The application of immunohistologicy staining allows the detection of infiltrating immune cells in malignant or infected tissues and is especially useful to characterize the cells at the vaccination site. Cells can be detected at the single cell level and their localization in the tissue environment can be determined but can be, at the best, semiquantitative and double- or multistaining is diffcult to establish. Download pdf-Format - T Cell Receptor Analysis
The T cell receptor analysis allows the analysis of the heterogeneity of T cells and their clonality and provides tools to follow the course of specific T cell clones during therapy, for example after adoptive T cell transfer in patients. Protocol for fixed cells: Download pdf-format, Protocol for unfixed cells: Download pdf-format
Innate Immune System
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Monocyte subpopulation quantification
This protocol describes the absolute quantification of monocyte subpopulations in peripheral human blood.
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Sputum sample processing
This protocol describes the steps that are necessary for the processing of sputum samples.
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Identification and characterization of regulatory T cells (Treg)
This protocol describes the characterization and identification of regulatory T cells using multicolor flow cytometry.
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Characterization of NK-cells
This protocol describes the subtyping of different NK-subpopulations using multicolor flow cytometry.
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Proliferation Assay
This protocol describes the steps that are necessary for staining cells with a cytoplasmic stain, so that the proliferation of the stained cells can be analyzed using multicolor flow cytometry.
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Professional immune monitoring encompasses the parallel application of different technologies, which together determine the frequency, phenotype, function and homing capacity of vaccination-induced lymphocytes in the circulation and within the targeted tissues. Only the combined use of the above mentioned techniques leads to a valid set of biomarkers as surrogates for successful immunotherapeutic strategies.
Some references for further reading:
Britten, C.M., Janetzki, S., van der Burg, S. H., Gouttefangeas, C., Hoos, A. Toward the harmonization of immune monitoring in clinical trials: Quo vadis? Cancer Immunol. Immunother. 57; 285-288 (2008)
Britten, C. M., Gouttefangeas, C., Welters, M. J. P., Pawelec, G., Koch, S., Ottensmeier, C., Mander, A., Walter, S., Paschen, A., Müller-Berghaus, J., Haas, I., Mackensen, A., Køllgaard, T., thor Straten, P., Schmitt, M., Giannopoulos, K., Maier, R., Veelken, H., Bertinetti, C., Konur, A., Huber, C., Stevanović, S., Wölfel, T., van der Burg, S. H. The CIMT-monitoring panel: a two-step approach to harmonize the enumeration of antigen-specific CD8+ T lymphocytes by structural and functional assays. Cancer Immunol. Immunother. 57; 289-302 (2008)
Janetzki, S., Panageas, K. S., Ben-Porat, L., Boyer, J., Britten, C. M., Clay, T. M., Kalos, M., Maecker, H. T., Romero, P., Yuan, J., Kast, M., Hoos, A. Results and harmonization guidelines from two large-scale international Elispot proficiency panels conducted by the Cancer Vaccine Consortium (CVC/SVI). Cancer Immunol. Immunother. 57: 303-315 (2008)
Keilholz, U., Martus, P., Scheibenbogen, C. Immune monitoring of T cell responses in Cancer. Clin. Cancer Res. 12; 2346-2352 (2006)
Knutson, K. L., dela Rosa, C., Disis, M. L. Laboratory analysis of T cell immunity. Frontiers in Biosciences 11; 1932-1944 (2006).
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