Heiko Steuer

Cell adhesion profiling using extracellular matrix protein microarrays.

We have developed a microarray-based system for cell adhesion profiling of large panels of cell-adhesive proteins to increase the throughput of in vitro cell adhesion assays, which are currently primarily performed in multiwell plates. Miniaturizing cell adhesion assays to an array format required the development of protocols for the reproducible microspotting of extracellular matrix (ECM) protein solutions and for the handling of cell suspensions during the assay. We generated ECM protein microarrays with high reproducibility in microspot protein content using nitrocellulose-coated glass microslides, combined with piezoelectric microspotting of protein solutions. Protocols were developed that allowed us to use 5000 cells or fewer on an array of 4 x 4 mm consisting of 64 microspots. Using this microarray system, we identified differences of adhesive properties of three cell lines to 14 different ECM proteins. Furthermore, the sensitivity and accuracy of the assays were increased using microarrays with ranges of ECM protein amounts. This microarray system will be particularly useful for extensive comparative cell adhesion profiling studies when only low amounts of adhesive substrate and cells, such as stem cells or cells from biopsies, are available.

A membrane‐bound FRET‐based caspase sensor for detection of apoptosis using fluorescence lifetime and total internal reflection microscopy

A caspase sensor based on Förster resonance energy transfer between fluorescent proteins is reported. Enhanced cyan fluorescent protein anchored to the inner leaflet of the plasma membrane of living cells is optically excited by an evanescent electromagnetic field and transfers its excitation energy via a spacer (DEVD) to an enhanced yellow fluorescent protein. Upon apoptosis, DEVD is cleaved and energy transfer is disrupted, as proven by pronounced changes in fluorescence spectra and decay times. Fluorescence spectroscopy and lifetime imaging (FLIM) is combined with total internal reflection fluorescence microscopy (TIRFM) for selective detection of this membrane‐bound caspase sensor. Fluorophores of the cytoplasm are thus excluded, and the signal‐to‐background ratio is increased considerably. In comparison with conventional or laser scanning microscopy, this permits long‐term observation of apoptosis in live cell cultures using very low absorption and avoiding light‐induced damages of the samples.

MicroPrep: Chip-based dielectrophoretic purification of mitochondria

We have developed a microfluidic system – microPrep – for subcellular fractionation of cell homogenates based on dielectrophoretic sorting. Separation of mitochondria isolated from a human lymphoblastoid cell line was monitored by fluorescence microscopy and further characterized by western blot analysis. Robust high throughput and continuous long‐term operation for up to 60 h of the microPrep chip system with complex biological samples became feasible as a result of a comprehensive set of technical measures: (i) coating of the inner surfaces of the chip with BSA, (ii) application of mechanical actuators to induce periodic flow patterns, (iii) efficient cooling of the device to ensure integrity of organelle, (iv) a wide channel to provide for high fluidic throughput, and (v) integration of a serial arrangement of 10 dielectrophoretic deflector units to enable separation of samples with a high particle load without clogging. Hence, microPrep yields tens of micrograms of enriched and purified mitochondria within hours. Western blots of mitochondria fractions showed that contaminating endoplasmatic reticulum was reduced by a factor 6 when compared with samples prepared by state of the art centrifugation.

Adhesion to Extracellular Matrix Proteins can Differentiate between Human Bone Marrow Derived Mesenchymal Stem Cells and Fibroblasts

Mesenchymal stem or stromal cells (MSC) contribute in vivo to wound repair and can be utilized for tissue regeneration. In contrast, fibroblasts may contribute to scar formation and may even hamper functional regeneration. Depending on the clinical application, MSC are sometimes attached to a scaffold to maintain the cells in the area of regeneration. We therefore screened for proteins that allow a preferential binding of MSC and avoid strong adherence of fibroblasts. The human MSC were isolated from bone marrow (bmMSC) or term placenta (pMSC). Synovial fibroblasts (SF) and dermal fibroblasts (DF) served as controls. In the first set of experiments, binding of bmMSC and SF to extracellular matrix (ECM) proteins was investigated by multiple substrate array (MSA®). From MSA® protein analyses 57 peptides with potential MSC-binding sites were selected and the binding of the cells to these peptides was determined. We report that MSC differ from fibroblasts in their binding to proteins of the extracellular matrix. MSC bind with higher efficiency to laminin-111, collagens-I, -III, and -IV and tenascin-C compared to fibroblasts, while both cell types bind with high efficiency to fibronectin, vitronectin, and laminin-511. We conclude that overall MSC seem less selective with respect to binding extracellular matrix components compared to fibroblasts, and fibroblasts attach to fewer proteins and peptides.

Förster resonance energy transfer-based total internal reflection fluorescence reader for apoptosis

A fluorescence reader for the detection of Forster resonance energy transfer (FRET) on surfaces of living cells is described. The method is based on multiple total internal reflections (TIR) of an incident laser beam within a glass slide, such that individual samples on top of the glass slide are illuminated simultaneously by an evanescent electromagnetic field. Enhanced cyan fluorescent protein (ECFP) anchored to the inner leaflet of the plasma membrane is optically excited and transfers its excitation energy via the peptide linker Asp-Glu-Val-Asp (DEVD) to an enhanced yellow fluorescent protein. Upon apoptosis, DEVD is cleaved, and energy transfer is disrupted, as proven by an increase of fluorescence intensity as well as of fluorescence lifetime of the donor ECFP. Due to selective excitation of membrane-associated fluorophores, intracellular fluorescence and background luminescence from the surrounding medium are eliminated. Therefore, this test system appears to be a sensitive device for the detection of apoptosis and more generally for drug screening or in vitro diagnosis on a nanometer scale.

Total internal reflection energy transfer (TIRET) microscopy for analysis of focal adhesions in living cells

Total internal reflection fluorescence microscopy (TIRFM) is used to measure non-radiative energy transfer between membrane associated proteins in living cells. Measurements are concentrated on focal contacts and their associated proteins focal adhesion kinase (FAK) and Paxillin (Pax) which play major roles with respect to cell migration, growth, and survival. These proteins are visualized after fusion with variants of green fluorescent protein (ECFP and EYFP), and an intermolecular energy transfer ECFP -> EYFP is deduced from fluorescence spectra as well as from fluorescence decay kinetics of single cells.

Fluorescence intensity, lifetime, and anisotropy screening of living cells based on total internal reflection techniques

A setup for fluorescence measurements of surfaces of biological samples, in particular the plasma membrane of living cells, is described. The method is based on splitting of a laser beam and multiple total internal reflections (TIR) within the bottom of a microtiter plate, such that up to 96 individual samples are illuminated simultaneously by an evanescent electromagnetic field. Two different screening procedures for the detection of fluorescence arising from the plasma membrane of living cells by High Throughput Screening (HTS) and High Content Screening (HCS), are distinguished. In the first case a rapid measurement of large sample numbers based on fluorescence intensity, and in the second case a high content of information from a single sample based on the parameters fluorescence lifetime (Fluorescence Lifetime Screening, FLiS) and fluorescence anisotropy (Fluorescence Lifetime Polarization Screening, FLiPS) is achieved. Both screening systems were validated using cultivated cells incubated with different fluorescent markers (e. g. NBD-cholesterol) as well as stably transfected cells expressing a fluorescent membrane-associating protein. In addition, particularly with regard of potential pharmaceutical applications, the kinetics of the intracellular translocation of a fluorescent protein kinase c fusion protein upon stimulation of the cells was determined. Further, a caspase sensor based on Förster Resonance Energy Transfer (FRET) between fluorescent proteins was tested. Enhanced cyan fluorescent protein (ECFP) anchored to the inner leaflet of the plasma membrane of living cells transfers its excitation energy via a spacer (DEVD) to an enhanced yellow fluorescent protein (EYFP). Upon apoptosis DEVD is cleaved, and energy transfer is disrupted, as proven by changes in fluorescence intensity and decay times.

A Membrane-Associated FRET Sensor for Detection of Apoptosis

A membrane associated caspase sensor based on Förster Resonance Energy Transfer (FRET) between enhanced cyan fluorescent protein (Mem-ECFP) and yellow fluorescent protein (EYFP) is reported. Upon apoptosis a caspase sensitive amino acid peptide linker (DEVD) between these proteins is cleaved, and pronounced changes of fluorescence spectra and lifetimes are observed. Membrane selective detection of fluorescent proteins in cultivated HeLa cervix carcinoma cells is achieved by total internal reflection fluorescence microscopy (TIRFM) with high sensitivity and resolution.