Analysis and sorting of live cells according to secreted molecules, relocated to a cell-surface affinity matrix.

Abstract

We have developed a technology for analysis and sorting of live cells according to secreted molecules. An artificial affinity matrix, specific for the secreted product of interest, is created on the cell surface, and the cells are allowed to secrete for a defined time period. The secreted molecules bind to the affinity matrix on the secreting cell and are subsequently labeled with specific fluorescent or magnetic staining reagents for cytometric analysis and cell sorting. Crossfeeding of the secreted products to other cells is prevented by decreasing the permeability of the incubation medium. This approach will have a wide range of applications in biotechnology and biomedical research. Here, we describe analysis and sorting of hybridoma cells, according to secreted antibodies, and of activated T lymphocytes, according to secreted cytokines.Proteins and other cellular products can be expressed either intracellularly, on the cell surface, or by secretion into the extracellular space. Despite its biological significance, only a few methods are presently available for the analysis of secreted products on the single cell level. This situation is mainly because secreted cellular products are hard to assign to the secreting cell, especially in a quantitative way. In the currently available protocols, the plaque assay (1), the ELISPOT assay (2), and the microdroplet technology (3), secreting cells are fixed in or on support matrices, which trap the secreted products for analysis. These approaches impose severe limitations because the secreted molecules still dissociate from the cell, and trapping matrix and cells must form a unit for further analysis and sorting of the cells. Here we describe another concept for cytometry and sorting of live cells based on secreted products. Basically, the secreted product is retained on the cell surface of the secreting cell, making it accessible to the powerful technologies for detection of surface markers. As shown schematically in Fig. 1, an affinity matrix for the secreted product is generated by attaching a specific antibody to the cell surface. Subsequently, the cells are allowed to secrete their products under defined conditions into a medium of low permeability for the secreted product. After removal of the cells from the incubation medium, they are stained for the secreted product, which is now bound to the cell-surface affinity matrix, with specific fluorochrome-, hapten-, or particle-labeled "detection" antibodies or other staining reagents. Preparation of Medium of Low Permeability. Sixty grams of gelatin (from bovine skin, 75 bloom, Sigma) was dissolved in 100 ml of warm PBS and dialyzed extensively, first against PBS and then against RPMI 1640 medium (GIBCO). For use, this stock solution was diluted (with RPMI medium/5% fetal calf serum) to a final concentration of 25% or 40% gelatin. MATERIAL AND METHODSEmbedding of Cells in Gelatin. A cell suspension of 106_107 cells in 100 ml of PBS/BSA was mixed with 1 ml of gelatinous medium (25 or 40% gelatin) prewarmed to 3...

Identification and isolation of murine antigen‐reactive T cells according to CD154 expression

“…Flexibility due to independence from certain MHC alleles and defined antigenic peptides is one of the major advantages of such functional assays. The analysis of specific T cells according to the expression of cytokines upon in vitro re-stimulation is currently the state-of-theart for the analysis of physiologic CD4 T cell responses [5][6][7]30]. The introduction of CD154 as a universal marker for all antigen-reactive CD4 T cells for the murine system will significantly improve these analyses.…”

1,25‐dihydroxyvitamin D3 promotes IL‐10 production in human B cells

“…IL-10 expression by B cells was analyzed also on the single-cell level, using the IL-10 capture assay ( [17,18,22] and Supporting Information Fig. 1).…”

“…Staining of viable IL-10-producing CD19 + B cells was performed after cell culture for 2-4 days and restimulation with 10 ng/mL PMA and 1 lg/mL ionomycin (both Sigma, Germany) by the IL-10 capture assay as previously described [17,18,22]. Briefly, the activated B cells were coated with a bivalent antibody matrix against CD45 and IL-10 (Miltenyi Biotec, Germany), which captures secreted IL-10 back on the surface of the secreting cell, where it can be detected with a secondary fluorescent IL-10 specific antibody (Supporting Information Fig.…”

Dichotomy of Autoreactive Th1 and Th2 Cell Responses to Desmoglein 3 in Patients with Pemphigus Vulgaris (PV) and Healthy Carriers of PV-Associated HLA Class II Alleles

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