Microplate live cell assay system for early events in mechanotransduction

For studying mechanotransduction in cultured cells, we developed a microplate assay using a fluorescence/luminescence plate reader equipped with software-controlled injectors to deliver a reproducible mechanical stimulus (adjustable for both timing and force) and immediately measure adenosine 5(')-triphosphate (ATP) release and calcium mobilization. Suspension or adherent chondrocyte cultures in 96-well plates were incubated with firefly luciferase and luciferin for the ATP assay or loaded with Fluo-3-acetoxy methylester for intracellular calcium measurement. Steady state ATP release was measured in resting cells; then mechanical stimulation was delivered by injection of an equal volume of buffer into the wells. Serial integrations of 20 to 500ms allowed real-time analysis of the time course of ATP release. Luminescence increased within 500ms indicating the rapidity of ATP release in chondrocyte mechanotransduction. Subsequent injection of a cell lysis solution allowed quantitation of total cellular ATP as an internal control of cell viability and number. Intracellular calcium was also elevated within 500ms of fluid injection. This assay is easily adapted for changes in intracellular pH or other ions by use of different commercially available fluorescent indicators. The live-cell assay using fluid injection as a mechanical stimulus is a valuable tool for dissecting the role of signaling pathways in mechanotransduction.     

Comparison of the three optical platforms for measurement of cellular respiration

We compared three optical platforms for measurement of cellular respiration: absolute oxygen consumption rates (OCRs) in hermetically sealed microcuvettes, relative OCRs measured in a 96-well plate with oil seal, and steady-state oxygenation of cells in an open 96-well plate. Using mouse embryonic fibroblasts cell line, the phosphorescent intracellular O₂ probe MitoXpress-Intra, and time-resolved fluorescence reader, we determined algorithms for conversion of relative OCRs and cell oxygenation into absolute OCRs, thereby allowing simple high-throughput measurement of absolute OCR values.     

Fast response temperature measurement and highly reproducible heating methods for 96-well plates

Hyperthermia, the procedure of exposing cells to a temperature between 42 degrees and 49 degrees C, has been shown to be a promising approach for cancer treatment. To understand the underlying mechanisms of hyperthermic killing of cancer cells, it is critical to have an accurate temperature measurement technique and a heating method with high reproducibility. To this end, we have developed a method using fine thermocouples with fast response time to measure the temperatures in multiple wells of a 96-well plate. The accuracy of temperature measurement was +/- 0.2 degree C. Such a capability allows a complete record of the time and temperature of the treatment procedure and helps define an accurate thermal dose. We have also compared several methods for heating 96-well plates and found that use of copper blocks in contact with the lower surface of the 96-well plate in an incubator provides a highly reproducible heating method. The common method of using water bath to heat cells in vitro resulted in a decrease of cell viability even at the control temperature of 37 degrees C and a decrease in the reproducibility of certain biological assays. In summary, using these improved techniques, proposed thermal dose can be defined more precisely, and highly reproducible heating in vitro can be achieved.     

Spatial distribution of the state of water in frozen mammalian cells

We describe direct determination of the state of intracellular water, measurement of the intercellular concentration of a cryoprotectant agent (dimethylsulfoxide), and the distribution of organic material in frozen mammalian cells. Confocal Raman microspectroscopy was utilized at cryogenic temperatures with single live cells to conduct high spatial resolution measurements (350 × 350 × 700 nm), which yielded two, we believe, novel observations: 1), intracellular ice formation during fast cooling (50°C/min) causes more pronounced intracellular dehydration than slow cooling (1°C/min); and 2), intracellular dimethylsulfoxide concentration is lower (by as much as 50%) during fast cooling, decreasing the propensity for intracellular vitrification. These observations have a very significant impact for developing successful biopreservation protocols for cells used for therapeutic purposes and for cellular biofluids.     

一种测定蛹虫草中虫草酸含量的酶标仪微量反应方法

研究和建立一种基于酶标仪-96孔板高通量测定虫草酸含量的检测方法,并对该方法进行性能评价。以酶标仪为检测仪器,在96孔板内按照设定反应条件微量加入样品和试剂进行显色反应,利用酶标仪测定吸光度值并计算虫草酸含量。通过检测精密度、重复性、回收率,并与分光光度计法进行比较,综合评价该方法的准确度、精确度。结果表明,测定数据具有较高的精密度(样品CM1的RSD值0.829%;样品CM2的RSD值1.772%)、重复性(标准样品B40的RSD值2.061%;样品CM2的RSD值1.599%)、回收率(平均回收率99.24%,RSD值3.666%),测定结果与分光光度法检测结果无显著差异(P>0.05)。结果表明,酶标仪微量法测定准确、重复性好,并可大大减少样品和试剂的用量,该方法方便、快捷、高效,可以替代分光光度法用于虫草酸含量的测定。     

Co-determination of ATP and proteins in Triton X 100 non-ionic detergent-opened monolayer cultured cells.

Human monolayer cells (HEp-2 and Hep G2) were cultured in 96-well plates. A modified Triton X 100 nonionic detergent extraction method was used for releasing intracellular ATP and protein in one step. The detergent technique was compared to perchloric acid (PCA) extraction. ATP was determined by the firefly bioluminescence method and ATP values were referred to cell protein (ATP:protein ratio). There was no significant difference in ATP data between detergent and PCA treatments. The ATP:protein ratio seems to be a sensitive tool for characterizing the metabolic activity of monolayer tissue culture cells. The protein-mobilizing capability of Triton X 100 depends on the type of cell culture used. Our modified extraction gives reliable ATP:protein values with one simple extraction step.     

Continuous,label-free, 96-well-based determination of cell migration using confluence measurement

Cellular migration is essential in diverse physiological and pathophysiological processes. Here, we present a protocol for quantitative analysis of migration using confluence detection allowing continuous, non-endpoint measurement with minimal hands-on time under cell incubator conditions. Applicability was tested using substances which enhance (EGF) or inhibit (cytochalasin D, ouabain) migration. Using a gap-closure assay we demonstrate that automated confluence detection monitors cellular migration in the 96-well microplate format. Quantification by % confluence, % cell free-area or % confluence in cell-free area against time, allows detailed analysis of cellular migration. The study describes a practicable approach for continuous, non-endpoint measurement of migration in 96-well microplates and for detailed data analysis, which allows for medium/high-throughput analysis of cellular migration in vitro.     

Fast filtration for metabolome sampling of suspended animal cells

A new method for sampling suspended animal cells by fast filtration is presented that allows rapid quenching of cellular metabolism and efficient separation of the cells from culture medium. Compared to sampling with a microstructure heat exchanger or centrifugation without prior quenching, the adenylate energy charge and the measured concentrations especially of metabolites with a high turnover rate or of metabolites early in metabolic pathways were substantially higher. No leakage of ATP from the cells was observed when using iso-osmotic NaCl solution in the washing step. The combination of fast filtration and cold methanol extraction is therefore suitable for intracellular metabolomic studies of suspended animal cell cultures and superior to other methods currently applied.     

Protocols and Programs for High-Throughput Growth and Aging Phenotyping in Yeast

In microorganisms, and more particularly in yeasts, a standard phenotyping approach consists in the analysis of fitness by growth rate determination in different conditions. One growth assay that combines high throughput with high resolution involves the generation of growth curves from 96-well plate microcultivations in thermostated and shaking plate readers. To push the throughput of this method to the next level, we have adapted it in this study to the use of 384-well plates. The values of the extracted growth parameters (lag time, doubling time and yield of biomass) correlated well between experiments carried out in 384-well plates as compared to 96-well plates or batch cultures, validating the higher-throughput approach for phenotypic screens. The method is not restricted to the use of the budding yeast Saccharomyces cerevisiae, as shown by consistent results for other species selected from the Hemiascomycete class. Furthermore, we used the 384-well plate microcultivations to develop and validate a higher-throughput assay for yeast Chronological Life Span (CLS), a parameter that is still commonly determined by a cumbersome method based on counting “Colony Forming Units”. To accelerate analysis of the large datasets generated by the described growth and aging assays, we developed the freely available software tools GATHODE and CATHODE. These tools allow for semi-automatic determination of growth parameters and CLS behavior from typical plate reader output files. The described protocols and programs will increase the time- and cost-efficiency of a number of yeast-based systems genetics experiments as well as various types of screens.     

On the noninvasive measurement of intracellular free magnesium by 31P NMR spectroscopy

We previously introduced a noninvasive measurement of the concentration of free Mg2+ in intact cells and tissues using 31P NMR. To resolve a controversy in the literature concerning the affinity of Mg2+ for ATP used in our procedure, the apparent dissociation constant of MgATP under simulated intracellular conditions has been determined by three independent magnetic resonance methods, including a newly developed combination procedure for determining this value at intracellular ATP levels. The new combination method, which utilizes 31P NMR to determine the degree of Mg2+ chelation of ATP and the dye antipyrylazo III for optical determination of free Mg2+, yielded a value of (50 +/- 10) microM for this apparent dissociation constant at pH 7.2 in the presence of 0.15 M K+ and 25 degrees C. We further show that hydroxyquinolines are not satisfactory indicators for optical determination of the Mg2+-nucleotide dissociation constant. From our determinations a low value of free Mg2+ (less than 1 mM) is established for all of the tissues studied, including perfused heart muscle, contrary to a recent report in the literature. Saturating human erythrocytes with Mg2+ results in an alpha- and beta-phosphorus resonance separation for intracellular ATP that is indistinguishable from that observed in a noncellular MgATP control under similar conditions, showing that MgATP resonances in this cell are unaffected by the cellular environment.     

Continuous flow method for extraction and bioluminescence assay of ATP in baker's yeast

Summary The intracellular ATP of baker's yeast (Saccharomyces cerevisiae) was measured using the bioluminescent firefly luciferase assay. Benzalkonium chloride and trichloro-acetic acid served in the experiments as extracting agents and optimal conditions for the extraction and assay of the intracellular ATP are reported. Using the results obtained from manually performed experiments two continuous flow systems were designed for the measurement of ATP in yeast cells during cell growth. Good correlation between the amount of cellular ATP and cell growth was found during the exponential growth phase.     

Sulforhodamine B colorimetric assay for cytotoxicity screening

The sulforhodamine B (SRB) assay is used for cell density determination, based on the measurement of cellular protein content. The method described here has been optimized for the toxicity screening of compounds to adherent cells in a 96-well format. After an incubation period, cell monolayers are fixed with 10% (wt/vol) trichloroacetic acid and stained for 30 min, after which the excess dye is removed by washing repeatedly with 1% (vol/vol) acetic acid. The protein-bound dye is dissolved in 10 mM Tris base solution for OD determination at 510 nm using a microplate reader. The results are linear over a 20-fold range of cell numbers and the sensitivity is comparable to those of fluorometric methods. The method not only allows a large number of samples to be tested within a few days, but also requires only simple equipment and inexpensive reagents. The SRB assay is therefore an efficient and highly cost-effective method for screening.     

Rapid Screening of HIV Reverse Transcriptase and Integrase Inhibitors

Although a number of anti HIV drugs have been approved, there are still problems with toxicity and drug resistance. This demonstrates a need to identify new compounds that can inhibit infection by the common drug resistant HIV-1 strains with minimal toxicity. Here we describe an efficient assay that can be used to rapidly determine the cellular cytotoxicity and efficacy of a compound against WT and mutant viral strains.The desired target cell line is seeded in a 96-well plate and, after a 24 hr incubation, serially dilutions of the compounds to be tested are added. No further manipulations are necessary for cellular cytotoxicity assays; for anti HIV assays a predetermined amount of either a WT or drug resistant HIV-1 vector that expresses luciferase is added to the cells. Cytotoxicity is measured by using an ATP dependent luminescence assay and the impact of the compounds on infectivity is measured by determining the amount of luciferase in the presence or the absence of the putative inhibitors.This screening assay takes 4 days to complete and multiple compounds can be screened in parallel. Compounds are screened in triplicate and the data are normalized to the infectivity/ATP levels in absence of target compounds. This technique provides a quick and accurate measurement of the efficacy and toxicity of potential anti HIV compounds.     

High throughput metabolic stability screen for lead optimization in drug discovery

A high throughput approach for the determination of in vitro metabolic stability and metabolic profiles of drug candidates has been developed. This approach comprises the combination of a Biomek FX liquid handling system with 96-channel pipetting capability and a custom-designed 96-well format on-line incubator with efficient thermal conductivity. This combination facilitates automated reagent preparation, sample incubation, and sample purification for microsome stability studies. The overall process is both fast and accurate and meets the challenges of high throughput screening for drug discovery. A custom designed, user-friendly computer program has been incorporated for large-scale data processing and report generation. Several applications are discussed that implement this strategy for rapid selection of compounds in early drug discovery.     

Determination of N-acetyl retigabine in dog plasma by LC/MS/MS following off-line microElution 96-well solid phase extraction

A high throughput off-line microElution 96-well solid phase extraction (SPE) followed by liquid chromatography with tandem mass spectrometry (LC/MS/MS) quantification for the determination of N-acetyl retigabine in dog plasma has been developed and validated. The method involves the use of microElution 96-well SPE for the simultaneous extraction of N-acetyl retigabine and rapid removal of its N-glucuronide metabolite that has shown to be problematic due to its instability using other clean-up methods. The microElution SPE technology eliminates the need for post-extraction solvent evaporation and greatly reduces sample preparation time consequently improving assay efficiency.     

Large-scale screening assay to measure epidermal growth factor internalization

Recently, we showed that the internalization of the epidermal growth factor (EGF) receptor is inhibited by hydrogen peroxide (H(2)O(2)) in human fibroblasts. In order to test the effect of various stress conditions on receptor internalization and to test a variety of antioxidants in their capacity to prevent or reduce the H(2)O(2)-induced inhibition of internalization, a screening assay was developed to measure the internalization in 96-well plates. In this assay, cells are exposed to biotin-conjugated EGF and the amount of internalized EGF is detected with horseradish peroxidase-conjugated streptavidin. We show that the results obtained by this new assay are comparable with those from internalization studies performed with radioactive labeled EGF. Therefore, the cellular internalization assay as presented here is a reliable method to measure EGF receptor internalization. Moreover, because elaborate processing of the cells is not required, the assay is a relatively fast and inexpensive method to study ligand-induced internalization in 96-well plates and thereby is suitable for large-scale screening of compounds or conditions interfering with this internalization.     

A fluorescence microscopy based genetic screen to identify mutants altered for interactions with host cells

The study of microbial intracellular pathogenesis has benefited from the application of immunofluorescence microscopy to characterize interactions of the pathogen with host cells. Unfortunately, immunofluorescence microscopy is impractical for screening the large number of bacterial mutants necessary to represent the entire genome of the pathogen. Screening has been limited due to the lack of materials suitable for high-throughput processing (e.g. 96-well plates) that also possess the optical features needed for high resolution fluorescence microscopy. Recently marketed 96-well Special Optics (SO) plates provide both the 96-well template ideal for high-throughput analysis and optical features suitable for fluorescence microscopy. Until this work, mutants needed for the study of a fluorescence-based virulence phenotype could not be obtained by direct screening approaches. In this study, SO plates were used to examine 11520 individual Salmonella typhimurium MudJ mutants for the loss of the ability to disrupt host cell endocytic compartments. The direct application of the fluorescence phenotype for screening allowed us to obtain a set of mutants to characterize the formation of lysosomal membrane glycoprotein (lgp) containing tubules upon Salmonella infection of HeLa epithelial cells. This approach will facilitate the characterization of a wide range of microbial phenotypes detectable by fluorescence microscopy.     

应用改良庆大霉素保护试验检测肠炎沙门氏菌侵袭力表型

肠炎血清型沙门氏菌 (Salmonella enterica serovar Enteritidis,SE) 是引起肠炎和全身感染重要的沙门氏菌血清型之一,了解沙门氏菌侵袭力表型对阐明其感染致病机制至关重要。传统庆大霉素保护试验 (GPA) 存在通量低、重复性差等缺点。文中利用96孔细胞培养和多孔道移液的高通量优势,结合菌落分区微量滴板计数法改良了传统GPA试验方案。应用改良的GPA方法检测了16株SE菌株对非吞噬细胞 (HT-29) 的入侵表型和43株SE菌株对吞噬细胞 (RAW264.7) 的胞内复制表型。通过比较分析SE强、弱菌株JL228和LN248对吞噬细胞 (RAW264.7) 的侵袭力表型发现,改良的GPA得出的数据组内和组间变异系数低、数据重复性强,胞内复制表型也与显微观察结果相符。通过实践应用发现,改良的GPA方法具有通量高、重复性强、结果可靠兼具省时、省力等优点,可作为沙门氏菌菌株侵袭力表型检测的更新方案,为进一步阐明其致病机制提供了更科学有效的方法。     

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.     

Adaptation of aequorin functional assay to high throughput screening

AequoScreen, a cellular aequorin-based functional assay, has been optimized for luminescent high-throughput screening (HTS) of G protein-coupled receptor (GPCRs). AequoScreen is a homogeneous assay in which the cells are loaded with the apoaequorin cofactor coelenterazine, diluted in assay buffer, and injected into plates containing the samples to be tested. A flash of light is emitted following the calcium increase resulting from the activation of the GPCR by the sample. Here we have validated a new plate reader, the Hamamatsu Photonics FDSS6000, for HTS in 96- and 384-well plates with CHO-K1 cells stably coexpressing mitochondrial apoaequorin and different GPCRs (AequoScreen cell lines). The acquisition time, plate type, and cell number per well have been optimized to obtain concentration-response curves with 4000 cells/well in 384-well plates and a high signal:background ratio. The FDSS6000 and AequoScreen cell lines allow reading of twenty 96- or 384-well plates in 1 h with Z' values of 0.71 and 0.78, respectively. These results bring new insights to functional assays, and therefore reinforce the interest in aequorin-based assays in a HTS environment.