A comprehensive analysis of intestinal microbiota by multi-parameter flow cytometry
German Rheumatism Research Centre Berlin - A Leibniz Insitute, Germany
The intestinal bacterial flora, or microbiota, is associated to a variety of diseases including cancer, neurological diseases, and autoimmune diseases. Analyses of stool samples of patients showed that in such diseases the gut microbiota composition is altered, a condition termed dysbiosis. So far, dysbiosis is mainly detected via 16 S rDNA sequencing and manifests as altered abundancy of bacterial phyla, class, family or even certain genera. Albeit the determination of altered bacterial composition is highly relevant, any information on the condition of the microbial community is missing. Cellular properties of the bacteria and immunoglobulin-mediated immune responses shaping the microbiota are relevant parameters to reconstruct the host-microbe interaction. To achieve a more comprehensive understanding of the human intestinal microbiota, we apply multi-parameter flow cytometry revealing the complexity of the bacterial community through quantitative DNA label and light scatter determination, analysis of endogenous coating of bacteria with IgA1, IgA2, IgM and IgG and different lectin-binding sugar moieties exposed on the bacterial cell surface. As such we have characterized healthy human stool samples as proof-of-concept for the feasibility and strength of microbiota flow cytometry to depict the complexity and individuality of the intestinal microbiota.
Characterization of chemical-specific CD4+ T cells in human blood
1Allergy Study Centre, German Federal Institute for Risk Assessment, Berlin, Germany; 2Department of Chemical and Product Safety, German Federal Institute for Risk Assessment, Berlin, Germany; 3Clinics of Dermatology and Venerology, Vivantes Klinikum Neukölln, Berlin, Germany
Allergic contact dermatitis (ACD) is a widespread skin condition mediated by T cells (delayed type IV hypersensitivity reaction). It occurs in response to exogenous agents and external stimuli (Militello et al., 2020) and clinically manifests with a dermatitis or “eczema”. Although it is estimated that about 30% of chemicals may induce an ACD reaction prediction of the sensitizing potential remains difficult. At present, no validated in vitro T cell-based tests are available.
Recently, we described a CD154 (CD40L) upregulation assay to detect nickel-specific CD4+ T cells in human blood (Aparicio-Soto et al., 2020). CD154 is an activation marker transiently expressed by specific naïve and memory CD4+ T cells shortly after antigen stimulation. We here adapted the CD154-upregulation assay to detect human non-metal chemical specific CD4+ T cells. As model allergen, we use 2,4,6-trinitrobenzenesulfonic acid (TNBS), which possesses extreme sensitizing capacity (Gerberick et al., 1992).
Modification of peripheral blood mononuclear cells (PBMC) with TNBS induced the generation of T cell epitopes. After CFSE labeling, ‘modified’ cells were co-cultured for 5 hours with unmodified ‘responder’ cells. We monitored the toxicity of different TNBS concentrations in both ‘modified’ and ‘responder’ cells, as well as the upregulation and co-expression of different activation markers (e.g. CD154, CD69, CD137) by the ‘responder’ cells. The highest frequencies of CD154+ CD4+ memory T cells were observed with 3 mM TNBS (mean ~0.05%). On average 80% of these cells co-expressed CD69. This finding and specific restimulation of TNBS-specific single cell clones confirmed TCR-mediated activation.
Summarizing, CD4+ T cell activation by organic contact allergen-induced epitopes may be detected with the CD154 upregulation assay. In the future, this approach may be optimized to in vitro test, diagnose and eventually predict the sensitization ability of other contact allergens.
Deeper insights in neutrophil metabolism by systematic enzyme mapping based on phasor-analyzed label-free NAD(P)H-FLIM
1Biophysical Analytics, German Rheumatism Research Center (DRFZ) , Berlin; 2Institute for Neuropathology, Charité – Universitätsmedizin, Berlin; 3Immune Dynamics, German Rheumatism Research Center (DRFZ), Berlin; 4Dynamic and Functional in vivo Imaging, Free University, Berlin
The ubiquitous co-enzymes NAD+ (oxidized)/ NADH (reduced) and their phosphorized variants NADP+/NADPH form the basis of the cellular energy metabolism including glycolysis and OxPhos as well as of pathogen-defense provided by NADPH oxidases (NOX). Since the oxidized forms are autofluorescent, they can serve as markers to monitor these life-sustaining mechanisms label-free in vivo. In contrast to their emission wavelength, their fluorescence lifetime changes with the enzyme to which they bind to.
We performed NAD(P)H fluorescence lifetime imaging microscopy (FLIM) in isolated human neutrophils in a two-photon microscope (in time domain) and analyzed the data using the phasor approach [Digman, 2008]. Both the evaluation and interpretation of such measurements in a real biological environment are challenging, especially since the two-photon action cross-sections of NAD(P)H are comparatively low and the exact values of the enzyme-bound NAD(P)H lifetimes are controversially discussed in this field [Ranjit, 2019]. Here we measured the fluorescence lifetime of the eleven most abundant NAD(P)H-dependent enzymes in solution. In order to support a systematic interpretation of NAD(P)H-FLIM data, we developed an algorithm, which allocates each pixel of a FLIM-image to one of these enzymes by vector-analysis. In this way, discrete enzyme-maps as well as maps of their general metabolic activity are created. [Leben, 2019].
Applied to FLIM-images of phagocyting neutrophils, this method reveals deeper insights in their metabolism, which go beyond the NOX activation leading to the oxidative burst. In some cells we found a different group of enzymes activated than in most others. This could be a sign of activation, since most activated immune cells switch their metabolism form the efficient, but slow OxPhos to an upregulated faster glycolysis, to face an infection [Gaber, 2017].
In this way, we demonstrated the power of our systematic evaluation framework for NAD(P)H-FLIM in retrieving the complexity of metabolism in cells and tissues.
High-throughput cell and spheroid mechanics in virtual fluidic channels
1ZIK HIKE, University of Greifswald, Greifswald, Germany; 2University Medicine, Greifswald
Microfluidic techniques have proven to be of key importance for achieving high-throughput cell mechanical measurements. However, their design modifications require sophisticated cleanroom equipment. Here, we introduce virtual fluidic channels as a flexible and robust alternative to Poly-dimethylsiloxane (PDMS) chips. Virtual channels are liquid-bound flows that can be created in almost arbitrary fluidic systems, e.g. standard flow cytometer cuvettes, and tailored in three dimensions within seconds for rheological studies on a wide size range of biological samples. We show that cell deformation in narrow virtual channels inside micrometer-sized systems is mainly driven by shear stress .By contrast, cells inside virtual channels of a large cuvette or capillary are deformed by an interfacial normal stress originating from the liquid-liquid interface. We demonstrate that this liquid-liquid interface acts as a high-frequency liquid cantilever for probing cell rheology on a millisecond timescale. As a proof-ofprinciple experiment, cells are treated with the actin depolymerizing drug cytochalasin D. A significant reduction in elastic modulus is found compared to untreated cells. Our results highlight that Young's modulus of single cells exceeds the one of tissue by one order of magnitude. In summary, we show that virtual channels might offer the ability for high-throughput mechanical cell and tissue characterization in almost arbitrary geometries.
Integrated Cytomics platform for improved environmental monitoring of pollination, air and water quality, combining high throughput (imaging) cytometry and deep learning
1Helmholtz-Centre for Environmental Research - UFZ, Germany; 2German Centre for Integrative Biodiversity Research - iDiv, Germany
Pollination, good air and water quality as ecosystem services are crucial aspects of our daily life and directly related to our well-being. An impairment of these ecosystem services has dramatic consequences for health and quality of life, e.g. in form of loss of agricultural products, respiratory diseases or restricted drinking water supply. Regular monitoring of pollination, air and water quality is therefore extremely important and traditionally done via microscopy. Microscopic investigations are of high quality but come with several restrictions like taxonomic expert requirement, difficulties in providing standardized taxonomic knowledge and taking long time, limiting the samples which can be processed in total. A new integrative Cytomics platform has been established to address different aspects of environmental research, including pollen and phytoplankton research. The platform includes an ImageStream X Mk II, a FACS Aria II, an Accuri C6 and a Multisizer 4, allowing for rapid flow cytometric assarys, but also for detailed image-based analytics or sorting. The innovative combination of high throughput imaging flow cytometry and deep learning is suggested as a promising tool to enable faster analyses, by keeping the microscopic evaluation of samples.The methods which will be developed should directly inform scientist or local stake holders about aspects of pollination, as well as air and water quality. This will help to deliver early warning information for toxic organisms in drinking or bathing water, inform about relevance of certain pollinators or supports physicians with allergic pollen forecast.
Light scattering pulse shape analysis with multiple forward scatter detectors in flow cytometry
1German Rheumatism Research Centre Berlin (DRFZ) - Flow Cytometry Core Facility; 2APE Angewandte Physik und Elektronik GmbH; 3Max Planck Institute for Molecular Genetics (MPIMG) - Flow Cytometry Facility
In a flow cytometer, objects of interest (e.g. cells, bacteria, extracellular vesicles) passing the laser spot create a time-dependent scattering intensity signal, resulting in a signal pulse. In standard instruments, this pulse shape is normally reduced to three read-out values: height, area, and width. Consequently, valuable information can be lost since these three values hardly represent a sample’s complex light scattering behavior. Moreover, the scattered light is commonly detected within a large solid angle which inhibits angular resolution.
Both pulse shape analysis with time resolution and the detection of scattered light with improved angular resolution have already been discussed. However, neither have they been considered to be combined nor did they become standard methods.
We are combining both angular and temporal resolution of scattered light signals for flow cytometry. To that end, a flow cytometer setup was equipped with modified optics for angle-resolved detection of forward scattered light. In addition, custom-made signal acquisition electronics and software were employed to measure light scattering pulse shapes and acquire cell-specific information beyond height, area, and width signals.
With this setup, we analyzed cells from two cell lines (HEK and Jurkat). Based on the pulse shape analysis by means of wavelet decomposition and k-means clustering, we were able to distinguish between cells in different phases of the cell cycle (G1, S, G2/M) using the angle-resolved forward scatter signals. The identification of the cell cycle populations was confirmed by fluorescent staining (PI and BrdU-FITC) and analysis of samples that were sorted based on the fluorescent staining.
In-depth pulse shape analysis for flow cytometry data enables label-free analysis of cells and could help in increasing the number of accessible parameters. Future work aims at discovering further applications and the implementation of label-free cell sorting based on pulse shape analysis, i.e. pulse-shape activated cell sorting (PACS).
Limbostomy: Longitudinal Intravital Microendoscopy in Murine Osteotomies
1Biophysical Analytics, German Rheumatism Research Center (DRFZ), Berlin, Germany; 2Veterinary Medicine, Freie Universität Berlin, Berlin, Germany; 3Immune Dynamics, German Rheumatism Research Center (DRFZ), Berlin, Germany; 4Department of Rheumatology and Clinical Immunology, Charité – Universitätsmedizin, Berlin, Germany; 5RISystem AG, RISystem AG, Landquart, Switzerland; 6Tissue Morphogenesis, Max Planck Institute for Molecular Biomedicine, Münster, Germany; 7Julius Wolff Institute, Charité – Universitätsmedizin, Berlin, Germany
Dedicated interplay of immune cells, mesenchymal cells, and vasculature over the time course of regeneration is required for successful bone healing. In order to quantify the spatiotemporal aspects of the underlying cell processes, new imaging methods are needed.
We developed a method termed Limbostomy by combining LIMB - our intravital two-photon fluorescence microendoscopy approach - with osteotomy, to quantify parameters of successful endochondral bone regeneration.
The internal fixator plate stabilizes the bone and hosts a modular endoscope based on gradient refractive index (GRIN) lenses. To correct for intrinsic optical aberrations of the GRIN lenses, we designed an image post-processing algorithm: Image plane deformations as well as background- and noise effects on the image quality were minimized. We are now able to observe sub-cellular processes and quantitatively analyze dynamic angiogenesis, cell motility and - interactions during bone regeneration.
Utilizing a transgenic reporter mouse strain with nuclear-GFP and membrane-tdTomato under the Cadherin-5 promoter, we identified two distinct phases of vascularization after injury. In an initial rapid vessel-sprouting phase the field of view is pervaded within 3-4 days post-osteotomy. This is followed by a remodeling phase of the vessel network until the end of our observation time, 14 days post-surgery.
Hematopoiesis, cellular niches, immunological memory, vascularization and the underlying environmental and cellular interactions are only fully analyzable with approaches that allow fine-meshed continuous intravital time-lapse imaging with high-resolution in the bone marrow. Therefore, Limbostomy creates a unique set of opportunities to gain insights on spatiotemporal aspects of bone marrow biology during health and disease.
 J. Stefanowski, A.F. Fiedler, “Limbostomy: Longitudinal Intravital Microendoscopy in Murine Osteotomies.” Cytometry A (2020)
 D. Reismann, J. Stefanowski, “Longitudinal intravital imaging of the femoral bone marrow reveals plasticity within marrow vasculature.” Nature Communications (2017)
Longitudinal Intravital Multiphoton Microendoscopy Reveals Vascularization during Bone Regeneration To Occur in Two Distinct Phases and To Be Preceded by CX3CR1+ Myeloid Cells
1Deutsches Rheuma-Forschungszentrum, 10117 Berlin, Germany; 2Department of Rheumatology and Clinical Immunology, Charité - Universitätsmedizin, 10117 Berlin, Germany; 3Berlin-Brandenburg Center for Regenerative Therapies, Charité - Universitätsmedizin, 10117 Berlin, Germany; 4Dynamic and Functional in vivo imaging, Department of Veterinary Medicine, Freie Universität Berlin, Germany
Successful vascularization and osteoimmunological interactions are essential for a good healing outcome after fractures. Absence of macrophages has been associated with delayed healing in long bone osteotomies. However, the dynamics of how the injury site is vascularized and the role of macrophage subsets remain elusive. Using longitudinal intravital multiphoton microendoscopy in murine osteotomies (Limbostomy) we observed two phases of vascularization shown in Cadherin-5 reporter mice: 1. An early vascularization at 3-4 days post-osteotomy, which was completed within 24 hours and 2. a continuous remodeling of the vascular network until 14 days post-surgery. Immunofluorescence histology of the bone marrow revealed the vasculature in both phases to resemble the immunophenotype of CD31+Endomucin+ endothelial cells (type H vessels) an endothelial cell type that is linked to bone formation. During early vascularization of the fracture hematoma is preceded by motile CX3CR1+ cells shown in CX3CR1GFP reporter mice. Furthermore, immunofluorescence histology showed CX3CR1+ cells to express the pan-macrophage marker F4/80 in the second phase of vascularization. Of all cells in the osteotomy gap, CX3CR1+F4/80+Gr-1- mononuclear phagocytes, which resemble the immunophenotype of non-classical monocytes, constitute the major population. These results suggest that non-classical monocytes actively participate in the vascularization process of type H vessels to support bone formation.
ROS induced cell mechanical alterations in suspension and adherent cells
1Biomechanics, ZIK-HIKE, Universität Greifswald, Greifswald, Germany; 2Universitätsmedizin Greifswald, Greifswald, Germany
Increase in oxidative stress has been linked to many haematological and neurological disorders. Reactive oxygen species (ROS) are one of the primary sources of oxidative stress which are associated with essential alterations in cell physiology1. Mechanical properties have long been established as a label-free biomarker, but their interplay with alternating levels of ROS has not been thoroughly investigated. This study focusses on understanding the impact of oxidative stress on the mechanical properties of the human leukaemia cell line (HL-60) and immortalized rat brain C6 glioma cells. In an in-vitro assay, for ROS was generated by exposing cells to varying concentrations of hydrogen peroxide. Using real-time fluorescence deformability cytometry2, we link for the first time the molecular phenotype of ROS using MitoSOX-red a fluorescent marker to changes in the mechanical phenotype which is a label-free biomarker. We show for micromolar concentrations of H2O2 induces different alterations in cell mechanical properties between both the cell types. For adherent cells, we find no changes in Young’s modulus, but for suspended cells we observe a different cell response to oxidative stress of increased elastic modulus. Alterations in Young’s modulus are not accompanied by significant changes in levels of microtubule and F-actin levels as detected by flow cytometry analysis but can be attributed to significant changes in cytoplasmic pH.
1. Liguori I, Russo G, Curcio F, et al. Oxidative stress, aging, and diseases. Clin Interv Aging. 2018. doi:10.2147/CIA.S158513
2. Rosendahl P, Plak K, Jacobi A, et al. Real-time fluorescence and deformability cytometry. Nat Methods. 2018. doi:10.1038/nmeth.4639
SARS-Cov-2 induces enhanced intestinal immune responses in severe COVID-19 patients
1German Rheumatism Research Center (DRFZ), a Leibniz Institute, Berlin 10117, Germany; 2Charité-Universitätsmedizin Berlin
The Coronavirus disease 2019 (COVID-19) pandemic is caused by the severe acute respiratory syndrome coronavirus 2 (SARS-Cov-2). Initial infection occurs through binding of the Receptor binding domain (RBD) of the viral Spike protein (S) to the ACE2 receptor, which is expressed in the nasopharynx, but also vastly on enterocytes of the gastrointestinal tract. It was shown that a SARS-Cov-2 infection could lead to gastrointestinal manifestations, raising the question how the mucosal immune response towards the virus is regulated. We analyzed fecal samples of intensive care unit (ICU) COVID-19 patients and could observe a highly disbiotic microbiota profile by flow cytometrie of bacterial populations. Furthermore, the commensal bacterial coating with IgA2 and IgG was enhanced in patients compared to healthy individuals, which correlated with the levels of unbound immunoglobulins in the fecal supernatants. Subsequently, we could determine that IgA2 and IgA1 fecal antibodies from COVID-19 patients are specific to the SARS-Cov-2 Spike protein. Thus, our data show that COVID-19 is associated with intestinal, mucosal immune response, which is characterized by an aberrant production of IgA2 and IgG antibodies.
Spatially resolved, in-depth cell profiling and quantification in human tissues by Multiplexed Histology and its biological applications
1Charité Universitätsmedizin Berlin, Germany; 2DRFZ Berlin, Germany; 3Freie Universität Berlin, Germany
The phenotype and function of cells are essentially linked to their tissue localization and to the interactions with the microenvironment. Based on that premise, we established a highly multiplexed immunofluorescence microcopy technique and combined it with a customized analysis pipeline that allows investigation of the overall tissue composition, retaining both spatial and single-cell resolution. Thereby, we have identified major immune cell populations in several organs, but also rare cell types, such as innate lymphoid cells (ILCs) and plasma cells, two cell types that we found to localize closely together in particular areas of the tonsils. We have identified new markers for ILC characterization, which were subsequently validated by RNA-seq, and we identified conserved stromal landmarks for ILC localization within and across tissues. We are currently studying local immune responses in several tissue pathologies, such as kidney tumors and transplants, colon samples from Crohn´s disease patients, as well as lungs and SLOs from COVID-19-deceased patients, in order to gain insights on the interrelation between immune cell types and structural tissue components, possibly mediating tissue inflammation and, ultimately, disease.
Stress-strain relation in virtual fluidic channels analyzed by particle image velocimetry
University Greifswald, Germany
Real-Time Deformability Cytometry (RT-DC) is a label-free technique for biomechanical phenotyping with high-throughput. However, in order to apply well-defined hydrodynamic stress on co-flowing cells dedicated channels with micrometer-precision are required. Recently, we demonstrated that virtual fluidic channels provide a versatile alternative to lithography based channels. Virtual fluidic channels are tunable liquid-liquid interfaces capable to constrain and deform cells that are created within a glass cannula. The cells mechanical properties can be deduced from a simple Kelvin-Voigt model, i.e. the proportionality between interfacial stress and cell strain. However, so far the virtual channel interfacial stress is poorly understood since it is highly connected to flow velocity distribution within the channel. To shed some light on the relation between flow profile and interfacial stress acting on cells we utilize micrometer resolution particle image velocimetry. Small tracer particles are introduced to the liquid and follow the flow profile faithfully. Observing the motion of the entrained tracer particles enables to calculate the flow velocity and direction both inside and outside the virtual fluidic channel. The flow profile of a virtual fluidic channel is characterized by a steep velocity gradient in close vicinity to the liquid-liquid interface. Linking cell deformation with flow velocity gradients promises a deeper understanding between the mechanical stress distribution on the cell surface and the consequential cell strain.
Superposition of viscosity maps and metabolism in parasitically infested intestinal tissue using BODIPY and NAD(P)H fluorecence lifetimes
1Biophysical Analytics, DRFZ - A Leibniz Institute, Berlin; Dynamic and functional in vivo imaging, Veterinary Medicine, Freie Universität, Berlin; 2DRFZ - A Leibniz Institute, Berlin; 3Immunodynamics, DRFZ - A Leibniz Institute, Berlin; Immunodynamics and intravital microscopy, Charité, Berlin; 4Beuth School for Applied Sciences; 5Institute for Immunology, Veterinary Medicine, Freie Universität, Berlin
Previous findings suggest a strong correlation of interactions between viscosity changes of the mucus of parasitically infested intestine, its metabolism and interaction processes with the parasites (especially as far as the behavior of the co-enzymes NAD(P)H is concerned). Thereby, defense mechanisms of the host, e.g. based on the enzymes NOX2 and NOX4, as well as dynamic parasite migration within the intestine as a counter-reaction are expected to be highly relevant in this context. Combining these physical parameters with spatiotemporal resolution in tissue opens new perspectives on the metabolic behaviour of host and parasites.
The fluorescence lifetime of the lipofilic dye BODIPY varies with the viscosity of the medium in which it is dissolved. On the other hand, the fluorescence lifetime of NAD(P)H depends both on general metabolic activity and specific enzymatic function. Due to different emission spectra of BODIPY and NAD(P)H, we are able to synchroniously monitor by fluorescence lifetime imaging (FLIM) NAD(P)H-dependent metabolism and, based on BODIPY fluorescence, mucus viscosity, in one and the same ROI within murine intestine samples. Based on a calibration curve of BODIPY fluorescence lifetime as a function of viscosity, we were able to determine absolute, local changes in mucus viscosity. Together with NAD(P)H-FLIM, we created a metabolic and viscosity 3D map of healthy intestine tissue.We extended our investigations to duodenum of mice infected with Heligmosomoides polygyrus. We observed changes in viscosity in close proximity to the worms and were able to visualise fluid dynamic flow processes in the stained mucus while simultaneously recording the worm movements over time.
By using BODIPY and NAD(P)H-FLIM, we could show that we are able to gain reliable results in
measuring mucus-viscosity and NAD(P)H-based metabolic processes.
Testing the B cell surfaceome by a bar-coded cytometry assay
Albert-Ludwigs-Universität Freiburg, Germany
We are studying the nanoscale organization of receptors on the B cell surface and found that most molecules are highly organized at nano-distances. In our study we provided evidence of the functional relevance of this nanoscale membrane organization. Thus, different receptor systems on the lymphocyte surface are not residing in perfect isolation from, but are functionally connected to each other. To learn more about the functional association of receptors on the B lymphocyte we are using the human Burkitt lymphoma cell line Ramos that we render receptor deficient with the CRISPR/Cas9 technique. For our study of the alteration of proteins on the Ramos B cell surface before and after a CRISPR/Cas9-mediated gene deletion we developed a barcoded flow cytometry assay, which allows us to compare the surface protein expression pattern of up to 16 differently color-coded Ramos B cells. For this we expose different genetically modified Ramos cell lines to 4 increasing concentrations of CytoTell Blue and/or CytoTell Green. The cells are then mixed together and distributed over separate tubes for further staining with fluorescent antibodies each specific for a given B cell surface marker. This barcoding technique is then used to directly compare wild type Ramos B cells to either normal human B cells or to Ramos cell mutants. These studies demonstrate the of receptors on the B cell surface. We also use Ramos cells and the barcoding technique for the detection of antibodies directed against the SARS-CoV-2 spike protein in the blood of infected or vaccinated persons.
This project was supported by the Deutsche Forschungsgemeinschaft through the TRR130-P02 and R01 grant 1R01AI145656-01.
The role of surface contacts in 2D and 3D microenvironments for cell mechanical properties
1ZIK HIKE, University of Greifswald, Germany; 2University Medicine Greifswald, Germany
Cells form with their microenvironment a network of biological and physicochemical signals that stem from cell-cell and cell-matrix contacts. Several pathologies including oncological disorders are associated with changes in such contacts but a comparative investigation by different approaches substantiating their relevance towards cell mechanics has, to our knowledge, never been conducted.
Here, we examine the role played by the substrate for the mechanical properties of HEK293T cells grown in 2D monolayers and spheroids as a 3D cell culture model. Experiments are performed using real-time deformability cytometry (RT-DC) and atomic force microscopy (AFM) in comparative assays.
Our AFM results show that cells cultured in 2D have a Young’s modulus that is significantly higher than that of 3D cultured cells. Interestingly, when cells are detached from the 2D substrate or the 3D matrix and captured in suspension, they become considerably stiffer. Comparing our AFM data to RT-DC results, which probe cells in suspension, we observe the same increase in elastic modulus independent of cell culture geometry. Our findings suggest, that the mechanical phenotype of adherent cells is to a large extent dominated by the presence of a substrate and less by the dimensionality of the cell environment. Furthermore, we look into the molecular basis of cell mechanics by deducing the levels of cytoskeletal proteins such as β actin, α/β tubulin, vinculin and talin by fluorescent flowcytometry and western blotting.