Reliable, standardized measurements for cell mechanical properties.

Atomic force microscopy (AFM) has become indispensable for studying biological and medical samples. More than two decades of experiments have revealed that cancer cells are softer than healthy cells (for measured cells cultured on stiff substrates). The softness or, more precisely, the larger deformability of cancer cells, primarily independent of cancer types, could be used as a sensitive marker of pathological changes. The wide application of biomechanics in clinics would require designing instruments with specific calibration, data collection, and analysis procedures. For these reasons, such development is, at present, still very limited, hampering the clinical exploitation of mechanical measurements. Here, we propose a standardized operational protocol (SOP), developed within the EU ITN network Phys2BioMed, which allows the detection of the biomechanical properties of living cancer cells regardless of the nanoindentation instruments used (AFMs and other indenters) and the laboratory involved in the research. We standardized the cell cultures, AFM calibration, measurements, and data analysis. This effort resulted in a step-by-step SOP for cell cultures, instrument calibration, measurements, and data analysis, leading to the concordance of the results (Young's modulus) measured among the six EU laboratories involved. Our results highlight the importance of the SOP in obtaining a reproducible mechanical characterization of cancer cells and paving the way toward exploiting biomechanics for diagnostic purposes in clinics.

Uptake of platelets by cancer cells and recycling of the platelet protein CD42a.

BACKGROUND: It is well accepted that the bidirectional crosstalk between platelets and cancer cells promotes tumorigenesis and metastasis. In an early step, cancer cells trigger platelet granule and extracellular vesicle release that is needed to facilitate cancer cell survival in circulation. OBJECTIVES: To discover the early crosstalk of cancer cells and platelets. METHODS: Cancer cells were incubated with freshly isolated and stained human platelets. Confocal laser scanning microscopy and flow cytometry was used to visualize and to quantify platelet uptake and the membrane presence of CD42 on cancer cells. Dyngo4a was used to test if platelet uptake is a dynamin-dependent process. RESULTS: We found a dynamin-dependent uptake of platelets by cancer cells. This is followed by the recycling of the platelet-specific protein CD42a and its incorporation into cancer cells' plasma membrane, which is not a result of platelet RNA transfer by platelet-derived microparticles and exosomes. Time course of platelet uptake follows a sigmoid function revealing that 50% of the cancer cells are positive for platelets after approximately 38 min. Platelet uptake was observed for the tested cancerous cells (A549, MCF-7, and MV3) but not for the non-cancerous cell line 16HBE14o-. CONCLUSIONS: Our results demonstrate that cancer cells hijack platelets by phagocytosis and recycling of platelet membrane proteins. The uptake of platelets has additional advantages for cancer cells: access to the entire and undiluted platelet proteome, transcriptome, and secretome. These novel findings will allow further mechanistic elucidation and thus help us gain deeper insights into platelet-assisted hematogenous metastasis.

Quantification of heparin's antimetastatic effect by single-cell force spectroscopy.

In circulation, cancer cells induce platelet activation, leading to the formation of a cancer cell-encircling platelet cloak which facilitates each step of the metastatic cascade. Since cancer patients treated with the anticoagulant heparin showed reduced metastasis rates and improved survival, it is supposed that heparin suppresses the cloak's formation by inhibiting the interaction between platelet's adhesion molecule P-selectin with its ligands on cancer cells. To quantify this heparin effect, we developed a single-cell force spectroscopy approach and quantified the adhesion (maximum adhesion force [FA ] and detachment work [WD ]) between platelets and human non-small cell lung cancer cells (A549). A configuration was used in which A549 cells were glued to tipless cantilevers and force-distance (F-D) curves were recorded on a layer of activated platelets. The concentration-response relationship was determined for heparin at concentrations between 1 and 100 U/mL. Sigmoid dose-response fit revealed half-maximal inhibitory concentration (IC50 ) values of 8.01 U/mL (FA ) and 6.46 U/mL (WD ) and a maximum decrease of the adhesion by 37.5% (FA ) and 38.42% (WD ). The effect of heparin on P-selectin was tested using anti-P-selectin antibodies alone and in combination with heparin. Adding heparin after antibody treatment resulted in an additional reduction of 9.52% (FA ) and 7.12% (WD ). Together, we quantified heparin's antimetastatic effect and proved that it predominantly is related to the blockage of P-selectin. Our approach represents a valuable method to investigate the adhesion of platelets to cancer cells and the efficiency of substances to block this interaction.

Endothelial EphB4 maintains vascular integrity and transport function in adult heart.

The homeostasis of heart and other organs relies on the appropriate provision of nutrients and functional specialization of the local vasculature. Here, we have used mouse genetics, imaging and cell biology approaches to investigate how homeostasis in the adult heart is controlled by endothelial EphB4 and its ligand ephrin-B2, which are known regulators of vascular morphogenesis and arteriovenous differentiation during development. We show that inducible and endothelial cell-specific inactivation of Ephb4 in adult mice is compatible with survival, but leads to rupturing of cardiac capillaries, cardiomyocyte hypertrophy, and pathological cardiac remodeling. In contrast, EphB4 is not required for integrity and homeostasis of capillaries in skeletal muscle. Our analysis of mutant mice and cultured endothelial cells shows that EphB4 controls the function of caveolae, cell-cell adhesion under mechanical stress and lipid transport. We propose that EphB4 maintains critical functional properties of the adult cardiac vasculature and thereby prevents dilated cardiomyopathy-like defects.

Signals of the Neuropilin-1-MET Axis and Cues of Mechanical Force Exertion Converge to Elicit Inflammatory Activation in Coherent Endothelial Cells.

The neuropilin-1 (NRP1)-MET signaling axis regulates the motility of individual endothelial cells (ECs). It is unknown how this signaling pathway affects the endothelial barrier in coherent ECs forming a tight monolayer. We hypothesized that it is involved both in modulation of the endothelial barrier and in EC activation. To investigate the role of NRP1-MET signaling in inflammatory processes (e.g., systemic inflammatory response syndrome [SIRS] or snakebite-induced SIRS-like conditions), we employed the C-type lectin-related protein rhodocetin-αß (RCαß) as a specific trigger of this signal axis in ECs in vitro. In coherent HUVECs, RCαß reinforced the actin cytoskeleton and increased cell stiffness, thus favoring vascular endothelial cadherin-mediated transmission of intercellular forces. Increased cell stiffness was associated with enhanced activation of RhoA and nuclear translocation of NF-κB. Simultaneously, RCαß-triggered signaling via the NRP1-MET axis increased EC monolayer permeability, induced transcription of proinflammatory genes such as ICAM-1 and, consequently, leukocyte tethering. The RCαß-induced transcriptome differed from that induced by hepatocyte growth factor, although in both cases the same tyrosine kinase, MET, was involved. This was due to RCαß-mediated recruitment of the MET coreceptor NRP1 and additional Rho-mediated activation of the actomyosin system. RCαß induced similar transcriptional and cellular changes if external shear forces were applied. These data highlight the modulatory role of NRP1 as MET coreceptor, and they explain how some snake venoms induce SIRS-like conditions. Additionally, this study demonstrates that inflammatory activation of coherent ECs is triggered by converging signals that are induced by NRP1-MET signaling and influenced by intercellular forces.

The anti-adhesive effect of glycoclusters on Pseudomonas aeruginosa bacteria adhesion to epithelial cells studied by AFM single cell force spectroscopy.

The human opportunistic pathogen Pseudomonas aeruginosa (PA) is responsible for chronic infections of the respiratory epithelium in cystic fibrosis patients. PA takes advantage of an arsenal of virulence factors to infect and colonize human lungs. Among them, the lectin LecA favours epithelium invasion by interacting with host cell globotriaosylceramide (Gb3). A new therapeutic approach is based on the development of synthetic multivalent molecules (glycoclusters) targeting LecA with a higher affinity than its natural ligand. Atomic force microscopy-single cell force spectroscopy has been used to study the effect of glycoclusters on the bacteria-cell interaction. Glycoclusters have been shown to affect the detachment work and detachment force of the bacteria-cell interaction. The specificity and the efficiency of the glycocluster in targeting the lectin and destabilizing the PA-epithelial cell adhesion are demonstrated and discussed.

KCa3.1 channel inhibition leads to an ICAM-1 dependent increase of cell-cell adhesion between A549 lung cancer and HMEC-1 endothelial cells.

Early metastasis leads to poor prognosis of lung cancer patients, whose 5-year survival rate is only 15%. We could recently show that the Ca2+ sensitive K+ channel KCa3.1 promotes aggressive behavior of non-small cell lung cancer (NSCLC) cells and that it can serve as a prognostic marker in NSCLC. Since NSCLC patients die of metastases, we investigated whether KCa3.1 channels contribute to poor patient prognosis by regulating distinct steps of the metastatic cascade. We investigated the extravasation of NSCLC cells and focused on their adhesion to endothelial cells and on transendothelial migration. We quantified the adhesion forces between NSCLC cells and endothelial cells by applying single cell force spectroscopy, and we monitored transendothelial migration using live-cell imaging. Inhibition of KCa3.1 channels with senicapoc or KCa3.1 silencing increases the adhesion force of A549 lung cancer cells to human microvascular endothelial cells (HMEC-1). Western blotting, immunofluorescence staining and biotinylation assays indicate that the elevated adhesion force is due to increased expression of ICAM-1 in both cell lines when KCa3.1 channels are downregulated. Consistent with this interpretation, an anti-ICAM-1 blocking antibody abolishes the KCa3.1-dependent increase in adhesion. Senicapoc inhibits transendothelial migration of A549 cells by 50%. Selectively silencing KCa3.1 channels in either NSCLC or endothelial cells reveals that transendothelial migration depends predominantly on endothelial KCa3.1 channels. In conclusion, our findings disclose a novel function of KCa3.1 channels in cancer. KCa3.1 channels regulate ICAM-1 dependent cell-cell adhesion between endothelial and cancer cells that affects the transmigration step of the metastatic cascade.

PeakForce Tapping resolves individual microvilli on living cells.

Microvilli are a common structure found on epithelial cells that increase the apical surface thus enhancing the transmembrane transport capacity and also serve as one of the cell's mechanosensors. These structures are composed of microfilaments and cytoplasm, covered by plasma membrane. Epithelial cell function is usually coupled to the density of microvilli and its individual size illustrated by diseases, in which microvilli degradation causes malabsorption and diarrhea. Atomic force microscopy (AFM) has been widely used to study the topography and morphology of living cells. Visualizing soft and flexible structures such as microvilli on the apical surface of a live cell has been very challenging because the native microvilli structures are displaced and deformed by the interaction with the probe. PeakForce Tapping® is an AFM imaging mode, which allows reducing tip-sample interactions in time (microseconds) and controlling force in the low pico-Newton range. Data acquisition of this mode was optimized by using a newly developed PeakForce QNM-Live Cell probe, having a short cantilever with a 17-µm-long tip that minimizes hydrodynamic effects between the cantilever and the sample surface. In this paper, we have demonstrated for the first time the visualization of the microvilli on living kidney cells with AFM using PeakForce Tapping. The structures observed display a force dependence representing either the whole microvilli or just the tips of the microvilli layer. Together, PeakForce Tapping allows force control in the low pico-Newton range and enables the visualization of very soft and flexible structures on living cells under physiological conditions.

Cystic fibrosis transmembrane conductance regulator is involved in polyphenol-induced swelling of the endothelial glycocalyx.

Previous studies show that polyphenol-rich compounds can induce a swelling of the endothelial glycocalyx (eGC). Our goal was to reveal the mechanism behind the eGC-swelling. As polyphenols are potent modulators of fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel, the hypothesis was tested whether polyphenol-induced increase in CFTR activity is responsible for the eGC-swelling. The impact of the polyphenols resveratrol, (-)-epicatechin, and quercetin on nanomechanics of living endothelial GM7373 cells was monitored by AFM-nanoindentation. The tested polyphenols lead to eGC-swelling with a simultaneous decrease in cortical stiffness. EGC-swelling, but not the change in cortical stiffness, was prevented by the inhibition of CFTR. Polyphenol-induced eGC-swelling could be mimicked by cytochalasin D, an actin-depolymerizing agent. Thus, in the vascular endothelium, polyphenols induce eGC-swelling by softening cortical actin and activating CFTR. Our findings imply that CFTR plays an important role in the maintenance of vascular homeostasis and may explain the vasoprotective properties of polyphenols. FROM THE CLINICAL EDITOR: Many vascular problems clinically can be attributed to a dysregulation of endothelial glycocalyx (eGC). The underlying mechanism however remains unclear. In this article, the authors used nanoindentation and showed that polyphenols could swell the endothelial glycocalyx and alter its function. This investigative method can lead to further mechanistic studies of other molecular pathways.

ECM stiffness regulates glial migration in Drosophila and mammalian glioma models.

Cell migration is an important feature of glial cells. Here, we used the Drosophila eye disc to decipher the molecular network controlling glial migration. We stimulated glial motility by pan-glial PDGF receptor (PVR) activation and identified several genes acting downstream of PVR. Drosophila lox is a non-essential gene encoding a secreted protein that stiffens the extracellular matrix (ECM). Glial-specific knockdown of Integrin results in ECM softening. Moreover, we show that lox expression is regulated by Integrin signaling and vice versa, suggesting that a positive-feedback loop ensures a rigid ECM in the vicinity of migrating cells. The general implication of this model was tested in a mammalian glioma model, where a Lox-specific inhibitor unraveled a clear impact of ECM rigidity in glioma cell migration.

Paracellular transport through healthy and cystic fibrosis bronchial epithelial cell lines--do we have a proper model?

It has been reported recently that the cystic fibrosis transmembrane conductance regulator (CFTR) besides transcellular chloride transport, also controls the paracellular permeability of bronchial epithelium. The aim of this study was to test whether overexpressing wtCFTR solely regulates paracellular permeability of cell monolayers. To answer this question we used a CFBE41o- cell line transfected with wtCFTR or mutant F508del-CFTR and compered them with parental line and healthy 16HBE14o- cells. Transepithelial electrical resistance (TER) and paracellular fluorescein flux were measured under control and CFTR-stimulating conditions. CFTR stimulation significant decreased TER in 16HBE14o- and also in CFBE41o- cells transfected with wtCFTR. In contrast, TER increased upon stimulation in CFBE41o- cells and CFBE41o- cells transfected with F508del-CFTR. Under non-stimulated conditions, all four cell lines had similar paracellular fluorescein flux. Stimulation increased only the paracellular permeability of the 16HBE14o- cell monolayers. We observed that 16HBE14o- cells were significantly smaller and showed a different structure of cell-cell contacts than CFBE41o- and its overexpressing clones. Consequently, 16HBE14o- cells have about 80% more cell-cell contacts through which electrical current and solutes can leak. Also tight junction protein composition is different in 'healthy' 16HBE14o- cells compared to 'cystic fibrosis' CFBE41o- cells. We found that claudin-3 expression was considerably stronger in 16HBE14o- cells than in the three CFBE41o- cell clones and thus independent of the presence of functional CFTR. Together, CFBE41o- cell line transfection with wtCFTR modifies transcellular conductance, but not the paracellular permeability. We conclude that CFTR overexpression is not sufficient to fully reconstitute transport in CF bronchial epithelium. Hence, it is not recommended to use those cell lines to study CFTR-dependent epithelial transport.

Hyaluronan export through plasma membranes depends on concurrent K+ efflux by K(ir) channels.

Hyaluronan is synthesized within the cytoplasm and exported into the extracellular matrix through the cell membrane of fibroblasts by the MRP5 transporter. In order to meet the law of electroneutrality, a cation is required to neutralize the emerging negative hyaluronan charges. As we previously observed an inhibiting of hyaluronan export by inhibitors of K(+) channels, hyaluronan export was now analysed by simultaneously measuring membrane potential in the presence of drugs. This was done by both hyaluronan import into inside-out vesicles and by inhibition with antisense siRNA. Hyaluronan export from fibroblast was particularly inhibited by glibenclamide, ropivacain and BaCl(2) which all belong to ATP-sensitive inwardly-rectifying K(ir) channel inhibitors. Import of hyaluronan into vesicles was activated by 150 mM KCl and this activation was abolished by ATP. siRNA for the K(+) channels K(ir)3.4 and K(ir)6.2 inhibited hyaluronan export. Collectively, these results indicated that hyaluronan export depends on concurrent K(+) efflux.

Transient P2X7 receptor activation triggers macrophage death independent of Toll-like receptors 2 and 4, caspase-1, and pannexin-1 proteins.

The function of P2X(7) receptors (ATP-gated ion channels) in innate immune cells is unclear. In the setting of Toll-like receptor (TLR) stimulation, secondary activation of P2X(7) ion channels has been linked to pro-caspase-1 cleavage and cell death. Here we show that cell death is a surprisingly early triggered event. We show using live-cell imaging that transient (1-4 min) stimulation of mouse macrophages with high extracellular ATP ([ATP]e) triggers delayed (hours) cell death, indexed as DEVDase (caspase-3 and caspase-7) activity. Continuous or transient high [ATP]e did not induce cell death in P2X(7)-deficient (P2X(7)(-/-)) macrophages or neutrophils (in which P2X(7) could not be detected). Blocking sustained Ca(2+) influx, a signature of P2X(7) ligation, was highly protective, whereas no protection was conferred in macrophages lacking caspase-1 or TLR2 and TLR4. Furthermore, pannexin-1 (Panx1) deficiency had no effect on transient ATP-induced delayed cell death or ATP-induced Yo-Pro-1 uptake (an index of large pore pathway formation). Thus, "transient" P2X(7) receptor activation and Ca(2+) overload act as a death trigger for native mouse macrophages independent of Panx1 and pro-inflammatory caspase-1 and TLR signaling.

Recovery of ΔF508-CFTR function by analogs of hyaluronan disaccharide.

We recently discovered that hyaluronan was exported from fibroblasts by MRP5 and from epithelial cells by cystic fibrosis (CF) transmembrane conductance regulator (CFTR) that was known as a chloride channel. On this basis we developed membrane permeable analogs of hyaluronan disaccharide as new class of compounds to modify their efflux. We found substances that activated hyaluronan export from human breast cancer cells. The most active compound 2-(2-acetamido-3,5-dihydroxyphenoxy)-5-aminobenzoic acid (Hylout4) was tested for its influence on the activity of epithelial cells. It activated the ion efflux by normal and defective ΔF508-CFTR. It also enhanced the plasma membrane concentration of the ΔF508-CFTR protein and reduced the transepithelial resistance of epithelial cells. In human trials of healthy persons, it caused an opening of CFTR in the nasal epithelium. Thus compound Hylout4 is a corrector that recovered ΔF508-CFTR from intracellular degradation and activated its export function.

Paracellular permeability of bronchial epithelium is controlled by CFTR.

In normal airway epithelium, the cystic fibrosis transmembrane conductance regulator (CFTR) transports Cl(-) ions to the apical surface of the epithelium paralleled by the flow of water through transcellular and paracellular pathways. The hypothesis was tested whether CFTR not only regulates the transcellular but also the paracellular shunt pathway. Therefore, we performed measurements of transepithelial electrical resistance (TER) and paracellular (14)C-mannitol permeability in wtCFTR (16HBE14o(-)) and delF508-CFTR (CFBE41o(-)) expressing human bronchial epithelial cells. Under resting conditions, CFBE41o(-) cell monolayers exhibit a higher paracellular permeability and lower TER as compared to 16HBE14o(-) monolayers. Stimulation of CFTR by cAMP induces opposite effects in the two cell lines. 16HBE14o(-) monolayers show a sharp decrease of TER, in parallel with a concomitant increase of paracellular permeability. The change in paracellular permeability is mediated by a myosin II dependent mechanism because it can be blocked by the myosin light chain kinase inhibitor ML-7. In contrast, CFBE41o(-) cells respond to cAMP stimulation with a decrease of paracellular permeability, paralleled by slight increase of TER. We conclude that stimulation of wtCFTR increases vectorial transcellular salt transport and, simultaneously, the paracellular permeability allowing water to follow through the paracellular pathway. In contrast, in CF epithelium cAMP stimulation increases neither vectorial salt transport nor paracellular permeability which is likely to contribute to the CF pulmonary phenotype. Taken together, our results link CFTR dysfunction to an improper regulation of the paracellular transport route.

Normal and pathological erythrocytes studied by atomic force microscopy.

Erythrocytes (red blood cells, RBCs) are the most common type of blood cells in vertebrates. Many diseases and dysfunctions directly affect their structure and function. Employing the atomic force microscope (AFM) physical, chemical, and biological/physiological properties of RBCs can be studied even under near-physiological conditions. In this chapter, we present the application of different AFM techniques to investigate and compare normal and pathological RBCs. We give a detailed description for nondestructive immobilization of whole intact RBCs and explain preparation techniques for isolated native RBC membranes. High-resolution imaging of morphological details and pathological differences are demonstrated with healthy and systemic lupus erythematosus (SLE) erythrocytes revealing substructural changes due to SLE. We also present the technique of simultaneous topography and recognition imaging, which was used to map the distribution of cystic fibrosis transmembrane conductance regulator sites on erythrocyte membranes in healthy and cystic fibrosis-positive RBCs.

Formation of cysts by principal-like MDCK cells depends on the synergy of cAMP- and ATP-mediated fluid secretion.

It has been suggested that more than 70% of the renal cysts in patients with autosomal dominant polycystic kidney disease (ADPKD) arise from the collecting duct and that within this segment cysts originate almost exclusively from principal rather than intercalated cells. The mechanisms for this predisposition of principal cells have so far remained elusive. We, therefore, used Madin-Darby canine kidney (MDCK) subclones resembling principal cells and alpha-intercalated cells in a three-dimensional in vitro model to determine differences in cystogenesis and cyst growth, including the response to cyclic adenosine monophosphate (cAMP) elevation and the dependence on ATP signaling. We found that in vitro cysts developed only from principal-like but not from intercalated-like MDCK cell clones. This specificity could be verified in mixed MDCK cultures enriched for principal- or intercalated-like cells. In vitro cyst growth upon elevation of intracellular cAMP was mainly driven by fluid secretion, rather than increased cell proliferation. The cAMP-dependent fluid secretion was found to depend on extracellular adenosine-5'-triphosphate (ATP) and to act synergistically with purinergic signaling, as the use of the ATP scavenger apyrase, as well as the P2 receptor inhibitor suramin, reduced cAMP-driven fluid secretion, while increasing extracellular ATP potentiated cAMP-mediated cyst growth. In conclusion, we provide in vitro evidence for the ability of principal rather than intercalated cells to form cysts, based on a synergism of cAMP and ATP signaling in enhancing apical fluid secretion.

Real-time monitoring of cell elasticity reveals oscillating myosin activity.

The cytoskeleton is the physical and biochemical interface for a large variety of cellular processes. Its complex regulation machinery is involved upstream and downstream in various signaling pathways. The cytoskeleton determines the mechanical properties of a cell. Thus, cell elasticity could serve as a parameter reflecting the behavior of the system rather than reflecting the specific properties of isolated components. In this study, we used atomic force microscopy to perform real-time monitoring of cell elasticity unveiling cytoskeletal dynamics of living bronchial epithelial cells. In resting cells, we found a periodic activity of the cytoskeleton. Amplitude and frequency of this spontaneous oscillation were strongly affected by intracellular calcium. Experiments reveal that basal cell elasticity and superimposed elasticity oscillations are caused by the collective action of myosin motor proteins. We characterized the cell as a mechanically multilayered structure, and followed cytoskeletal dynamics in the different layers with high time resolution. In conclusion, the collective activities of the myosin motor proteins define overall mechanical cell dynamics, reflecting specific changes of the chemical and mechanical environment.

Myosin 1G (Myo1G) is a haematopoietic specific myosin that localises to the plasma membrane and regulates cell elasticity.

Immune cells navigate through different environments where they experience different mechanical forces. Responses to external forces are determined by the mechanical properties of a cell and they depend to a large extent on the actin-rich cell cortex. We report here that Myo1G, a previously uncharacterised member of class I myosins, is expressed specifically in haematopoietic tissues and cells. It is associated with the plasma membrane. This association is dependent on a conserved PH-domain-like myosin I tail homology motif and the head domain. However, the head domain does not need to be a functional motor. Knockdown of Myo1G in Jurkat cells decreased cell elasticity significantly. We propose that Myo1G regulates cell elasticity by deformations of the actin network at the cell cortex.

Elasticity measurement of living cells with an atomic force microscope: data acquisition and processing.

Elasticity of living cells is a parameter of increasing importance in cellular physiology, and the atomic force microscope is a suitable instrument to quantitatively measure it. The principle of an elasticity measurement is to physically indent a cell with a probe, to measure the applied force, and to process this force-indentation data using an appropriate model. It is crucial to know what extent the geometry of the indenting probe influences the result. Therefore, we indented living Chinese hamster ovary cells at 37 degrees C with sharp tips and colloidal probes (spherical particle tips) of different sizes and materials. We furthermore developed an implementation of the Hertz model, which simplifies the data processing. Our results show (a) that the size of the colloidal probe does not influence the result over a wide range (radii 0.5-26 microm) and (b) indenting cells with sharp tips results in higher Young's moduli ( approximately 1,300 Pa) than using colloidal probes ( approximately 400 Pa).