High-throughput and sensitive assay to measure yeast cell growth: a bench protocol for testing genotoxic agents

Intracellular metabolites and environmental agents continuously challenge the structural integrity of DNA. In the yeast Saccharomyces cerevisiae, the complete collection of open reading frame deletion mutants, in combination with powerful screening methods, allows for the comprehensive analyses of cellular responses to insult. We have developed a protocol to determine the sensitivity of growing yeast to DNA-damaging agents that is based on automatic measurements of the optical density of very small (100 microl) liquid cultures. This simple method is highly sensitive, provides quantifiable data and offers high-throughput screening capability. Starting with the treatment of cells with different doses of damaging agents, pre-prepared growing media containing 96-well plates are inoculated and cell population is automatically monitored every 10 min for 48 hours. With the aid of a multi-channel pipette, the sensitivity of a number of yeast strains to several concentrations of drug can be tested in triplicate in less then 4 hours.     

Towards a regional approach to cell mechanics

Here we review studies of the physical, material properties of animal cells and their cytoskeleton, such as elastic stiffness and fluid viscosity, that determine how they respond to, and are shaped by, forces inside and out. Currently and historically, most such studies have reported a single value for a cell property and/or propose a single broad structural model based on nonliving materials. We believe that such physical studies would be of more interest to most cell biologists if greater emphasis were placed on the well-established regional differences within a cell and the ability of the cell to quickly change its mechanical behaviors     

High-throughput analysis of HGF-stimulated cell scattering

Historically, only relatively low-throughput or expensive methods have been available to measure cell migration. Hepatocyte growth factor (HGF) is a ligand for the tyrosine kinase receptor Met that, in addition to mediating proliferation and survival, increases cell motility and metastasis. The authors have developed a high-throughput imaging assay for measuring inhibition of HGF-induced scattering in human HPAF-II pancreatic adenocarcinoma cells. Following treatment with test compounds and HGF for 24 h, cells are labeled with a nuclear stain and imaged at 10x magnification. The proximity of neighboring nuclei is measured, and the distribution of internuclear distances across each field of view is used to calculate the fraction of scattered cells. This method of analysis can be extended to other cell types and signaling pathways and, compared with other membrane-based migration assays currently available, the assay is significantly lower in cost, is less labor intensive, and provides higher throughput.     

Jet injection into polyacrylamide gels: investigation of jet injection mechanics

Jet injectors employ high-velocity liquid jets that penetrate into human skin and deposit drugs in the dermal or subdermal region. Although jet injectors have been marketed for a number of years, relatively little is known about the interactions of high-speed jets with soft materials such as skin. Using polyacrylamide gels as a model system, the mechanics of jet penetration, including the dependence of jet penetration on mechanical properties, was studied. Jets employed in a typical commercial injector, (orifice diameter: 152 microm, velocity: 170-180 m/s) were used to inject fluid into polyacrylamide gels possessing Young's moduli in the range of 0.06-0.77 MPa and hardness values in the range of 4-70 H(OO). Motion analysis of jet entry into polyacrylamide gels revealed that jet penetration can be divided into three distinct events: erosion, stagnation, and dispersion. During the erosion phase, the jet removed the gel at the impact site and led to the formation of a distinct cylindrical hole. Cessation of erosion induced a period of jet stagnation ( approximately 600 micros) characterized by constant penetration depth. This stage was followed by dispersion of the liquid into the gel. The dispersion took place by crack propagation and was nearly symmetrical with the exception of injections into 10% acrylamide (Young's modulus of 0.06 MPa). The penetration depth of the jets as well as the rate of erosion decreased with increasing Young's modulus. The mechanics of jet penetration into polyacrylamide gels provides an important tool for understanding jet injection into skin.     

High-throughput RNAi screening in vitro: from cell lines to primary cells

Small interfering RNAs (siRNAs) are being used to induce sequence-specific gene silencing in cultured cells to study mammalian gene function. Libraries of siRNAs targeting entire human gene classes can be used to identify genes with specific cellular functions. Here we describe high-throughput siRNA delivery methods to facilitate siRNA library screening experiments with both immortalized and primary cells. We adapted chemical reverse transfection for immortalized adherent cell lines in a 96-well format. The method is fast, robust, and exceptionally effective for many cell types. For primary cells and immortalized cells that are recalcitrant to lipofection-based methods, we developed electropermeabilization (electroporation) conditions that facilitate siRNA delivery to a broad range of cell types, including primary human T-cells, hMSC, NHA, NDHF-Neo, HUVEC, DI TNC1, RPTEC, PC12, and K562 cells. To enable high-throughput electropermeabilization of primary cells, we developed a novel 96-well electroporation device that provides highly efficient and reproducible delivery of siRNAs. The combination of high-throughput chemical reverse transfection and electroporation makes it possible to deliver libraries of siRNAs to virtually any cell type, enabling gene function analysis and discovery on a genome scale.     

High-throughput detection and multiplex identification of cell contaminations

Unnoticed cell culture contamination by viruses, Mycoplasma, or other cell lines is not uncommon and a threat to laboratory safety and the quality of scientific results. We developed and validated a novel high-throughput Multiplex cell Contamination Test (McCT), which is currently able to detect 37 contamination markers in a single reaction. The assay is based on multiplex PCR with target-specific primers and subsequent hybridization of amplimers to specific oligonucleotide probes. McCT proved to be highly specific, sensitive and robust, and allows to analyze more than 1000 cell lysates per week. In conclusion, the novel McCT assay is a powerful high-throughput tool in assessing cell line purity.     

High-throughput screening of cell lysates for ganglioside synthesis

A high-throughput screen to detect the synthesis of natural and non-natural gangliosides by cell lysates has been developed and automated. Utilizing the binding specificity of cholera toxin B-subunit for the oligosaccharide moiety of the ganglioside G_M1, the synthesis of sugar–sphingolipid glycosidic linkages was detected using a modified enzyme-linked immunosorbent assay (ELISA)/enzyme-linked lectin assay (ELLA). The screen was optimized and validated for high-throughput screening of cell lysates by evaluating different vectors, promoters, substrates and detection strategies. The extent of ganglioside synthesis was found to be proportional to enzyme concentration and length of incubation time. As a test of the finalized screen efficacy, individual colonies from a saturation mutagenesis library of nucleophile mutants of an endoglycoceramidase were screened to identify the most active enzyme for ganglioside synthesis. This screen should find general application in assaying both glycolipid biosynthesis and glycolipid hydrolysis, as it is highly sensitive and can be used with crude cell extracts.     

On tensegrity in cell mechanics

All models are wrong, but some are useful. This famous saying mirrors the situation in cell mechanics as well. It looks like no particular model of the cell deformability can be unconditionally preferred over others and different models reveal different aspects of the mechanical behavior of living cells. The purpose of the present work is to discuss the so-called tensegrity models of the cell cytoskeleton. It seems that the role of the cytoskeleton in the overall mechanical response of the cell was not appreciated until Donald Ingber put a strong emphasis on it. It was fortunate that Ingber linked the cytoskeletal structure to the fascinating art of tensegrity architecture. This link sparked interest and argument among biologists, physicists, mathematicians, and engineers. At some point the enthusiasm regarding tensegrity perhaps became overwhelming and as a reaction to that some skepticism built up. To demystify Ingber's ideas the present work aims at pinpointing the meaning of tensegrity and its role in our understanding of the importance of the cytoskeleton for the cell deformability and motility. It should be noted also that this paper emphasizes basic ideas rather than carefully follows the chronology of the development of tensegrity models. The latter can be found in the comprehensive review by Dimitrije Stamenovic (2006) to which the present work is complementary.     

Substrate mechanics and cell spreading

Cell spreading and cell locomotion arise from forces exerted by actin microfilaments upon the substratum. Using modified protein films at fluorocarbon oil--water interfaces as substrates, we have measured some minimal mechanical properties required of these films to support cell spreading forces in vitro. For murine 3T3-L1 fibroblasts, complete cell spreading was obtained when the films exceeded surface shear moduli and surface fracture points of 15 and 5 dyne/cm, respectively. The human WI-38 fibroblast required more robust films than did its transformed counterpart (WI-38/VA 13) in order to achieve equivalent spreading. These results are of significance in understanding the metastatic capabilities of cancer cells.     

A simple cell based assay to measure Parkin activity

Parkin is an ubiquitin-protein ligase mutated in Autosomal Recessive - Juvenile Parkinsonism. Here, we describe a cell-based assay to measure Parkin's ubiquitin-protein ligase activity. It relies on the ability of Parkin to recognise depolarised mitochondria and exploits a cell line where Parkin expression is inducible. In these cells, Parkin expression promotes mitophagy and accelerates cell death in response to mitochondrial depolarisers. Time-lapse imaging confirmed cell death and revealed increased perinuclear mitochondrial clustering following induction of Parkin expression in cells exposed to carbonyl cyanide m-chlorophenylhydrazone. Similar effects were not observed with α-synuclein or DJ-1, other proteins associated with the development of Parkinson's disease, confirming the specificity of the assay. We have used this assay to demonstrate that ligase-defective Parkin mutants are inactive, and cellular proteasomal activity (using the proteasomal inhibitors MG132, clasto-lactacystin β-lactone and epoxomicin) is essential for the Parkin mediated effect. As the assay is suitable for high-throughput screening, it has the potential to identify novel proteostasis compounds that stimulate the activity of Parkin mutants for therapeutic purposes, to identify modulators of kinase activities that impact on Parkin function, and to act as a functional read-out in reverse genetics screens aimed at identifying modifiers of Parkin function during mitophagy.     

High-throughput screening of bacteriorhodopsin mutants in whole cell pastes

Pre-term neonates and neonates in general exhibit physiological vitamin E deficiency and are at increased risk for the development of acute lung diseases. Apoptosis is a major cause of acute lung damage in alveolar type II cells. In this paper, we evaluated the hypothesis that vitamin E deficiency predisposes alveolar type II cells to apoptosis. Therefore, we measured markers of apoptosis in alveolar type II cells isolated from control rats, vitamin E deficient rats and deficient rats that were re-fed a vitamin E-enriched diet. Bax and cytosolic cytochrome c increased, and the mitochondrial transmembrane potential and Hsp25 expression was reduced in vitamin E deficiency. Furthermore, increased DNA-fragmentation and numbers of early and late apoptotic cells were seen, but caspases 3 and 8 activities and expression of Fas, Bcl-2, Bcl-x and p53 remained unchanged. Vitamin E depletion did not change the GSH/GSSG ratio and the activities of antioxidant enzymes. Thus, vitamin E deficiency may induce a reversible pro-apoptotic response in lung cells and sensitise them for additional insult. In agreement with this hypothesis, we demonstrate that in vivo hyperoxia alone does not induce apoptosis in type II cells of control rats but reversibly increases DNA-fragmentation and numbers of early apoptotic type II cells in vitamin E-depleted cells.     

High-throughput analysis of animal cell cultures using two-dimensional fluorometry

We report a new method which combines fluorescence spectroscopy at microtiter plate scale with multivariate statistical analysis for rapid and high-throughput analysis of secreted recombinant protein and viable cell growth in animal cell cultures. The potential of the method is demonstrated by application to cultures of three Chinese Hamster Ovary (CHO) cell clones with distinct IgG₄ antibody yields. Supernatant samples collected throughout culture time were analysed by two-dimensional fluorometry; significant changes were observed in the regions of tryptophan, metabolic cofactors and vitamins. Partial least squares regression was then used to correlate the entire fluorescence map with measured concentrations of antibody and viable cells. For both target variables, a model was calibrated with representative data from the two less productive clones and validated with data from the best producer clone; this allowed viable cell density to be predicted for the validation clone with an average error of 10%; even better, the secreted antibody could be predicted with an average error of 7%, proving the predictive capacity of the model beyond the calibration region. All the main spectral regions were required to establish the best correlations for both targeted variables. In conclusion, this method effectively analyzes cellular productivity in 96-well plate format, shortening the time spent in early phases of bioprocess development.     

High-throughput screening of bacteriorhodopsin mutants in whole cell pastes

A high-throughput screening method has been developed which enables functional analysis of bacteriorhodpsin in whole cell pastes. Reflectance spectra, from as little as 5 ml of Halobacterium salinarum cells, show close correspondence to that obtained from the purified purple membrane (PM), containing bacteriorhodopsin (BR) as the sole protein component. We demonstrate accurate quantification of BR accumulation by ratiometric analysis of BR (A_max 568) and a membrane-bound cytochrome (A_max 410). In addition, ground-state light- and dark-adapted (LA and DA, respectively) spectral differences were determined with high accuracy and precision. Using cells expressing the BR mutant D85N, we monitored transitions between intermediate-state homologues of the reprotonation phase of the light-activated proton pumping mechanism. We demonstrate that phenotypes of three mutants (D85N/T170C, D85N/D96N, and D85N/R82Q) previously characterized for their effect on photocycle transitions are reproduced in the whole cell samples. D85N/T170C stabilizes accumulation of the N state while D85N/D96N accumulates no N state. D85N/R82Q was found to have perturbed the pK_a of M accumulation. These studies illustrate the correspondence between pH-dependent ground-state transitions accessed by D85N and the transitions accessed by the wild-type protein following photoexcitation. We demonstrate that whole cell reflectance spectroscopy can be used to efficiently characterize the large numbers of mutants generated by engineering strategies that exploit saturation mutagenesis.     

High-throughput, nontargeted metabolite fingerprinting using nominal mass flow injection electrospray mass spectrometry

Flow injection electrospray-mass spectrometry (FIE-MS) is finding utility as a first-pass metabolite fingerprinting tool in many research fields. We provide a protocol that has proved reliable in large-scale research projects involving diverse sample matrices originating from plants, microbes and mammalian biofluids. Using Brachypodium leaf material as an example matrix all steps are summarized from sample extraction to data quality assessment. Alternative procedures for dealing with other common matrices such as bloods and urine are included. With little sample pretreatment, no chromatography and instrument cycle times of <5 min it is feasible to analyze >1,000 samples per week. Analysis of a typical batch of 240 samples (including first-pass data analysis) can be accomplished within 48 h.     

Kinetics and mechanics of cell adhesion

Cell adhesion is mediated by specific interaction between receptors and ligands. Such interaction provides not only physical linkage but also communication between the cell and its environment. The kinetics and mechanics of cell adhesion are coupled, because force can influence the formation and dissociation of receptor-ligand bonds. The kinetic rates and their force dependence determine how likely, how rapidly and how strongly cells bind as well as how long they remain bound. Since adhesion molecules are linked to apposing cellular membranes, their interaction is governed by two-dimensional (2D) kinetics. This is in contrast to the three-dimensional (3D) binding of soluble ligands to cell surface receptors. Unlike the 3D case in which many methods are available for measuring kinetic rates, not until recently have the 2D kinetic rates become experimentally measurable. In this review, I will discuss the recent progress in the experimental methods that enable quantification of the relevant kinetic and mechanical parameters, the fundamental concepts that underlie the physics of the biological phenomena, and the mathematical models that relate functions to the intrinsic properties of the adhesion molecules.     

Mesenchymal stem cell mechanics from the attached to the suspended state

Human mesenchymal stem cells (hMSCs) are therapeutically useful cells that are typically expanded in vitro on stiff substrata before reimplantation. Here we explore MSC mechanical and structural changes via atomic force microscopy and optical stretching during extended passaging, and we demonstrate that cytoskeletal organization and mechanical stiffness of attached MSC populations are strongly modulated over >15 population doublings in vitro. Cytoskeletal actin networks exhibit significant coarsening, attendant with decreasing average mechanical compliance and differentiation potential of these cells, although expression of molecular surface markers does not significantly decline. These mechanical changes are not observed in the suspended state, indicating that the changes manifest themselves as alterations in stress fiber arrangement rather than cortical cytoskeleton arrangement. Additionally, optical stretching is capable of investigating a previously unquantified structural transition: remodeling-induced stiffening over tens of minutes after adherent cells are suspended. Finally, we find that optically stretched hMSCs exhibit power-law rheology during both loading and recovery; this evidence appears to be the first to originate from a biophysical measurement technique not involving cell-probe or cell-substratum contact. Together, these quantitative assessments of attached and suspended MSCs define the extremes of the extracellular environment while probing intracellular mechanisms that contribute to cell mechanical response.