Rapid, fluorometric DNA determination for chick limb-bud mesenchymal-cell microcultures

Summary Micromass cultures of chick and mouse limb-bud mesenchymal cells are commonly used for in vitro studies of cellular differentiation. Previously, adaptation of these cultures to 96-well plates facilitated analyses of various aspects of cellular behavior and the effects of different media components in these cultures. These adjustments allowed development of a serum-free medium for chick limb-bud mesenchymal cells and substantially decreased costs associated with media and reagents. Here we report a further development for this model system; a Hoechst 33342-based in situ DNA assay that provides reliable data much more quickly and with considerably less effort than had been feasible in the past. Because it allows quantitation of products of cellular differentiation and DNA in the same cultures, the number of cultures needed to provide the same data is essentially halved and the accuracy of normalized values for quantitative estimates of markers of differentiation is improved. Studies of the effects of retinoic acid on chick limb-bud mesenchymal cells were performed to document the usefulness of this method.     

Rapid detection of mycoplasma contamination in cell cultures using sybr green-based real-time polymerase chain reaction

Summary We have developed a simple method for rapid detection of mycoplasma contamination in cell cultures using SYBR Green-based real-time polymerase chain reaction (PCR). To detect eight common contaminant mollicutes, including Mycoplasma (M. arginini, M. fermentans, M. orale, M. hyorhinis, M. hominis, M. salivarium, M. pirum) and Acholeplasma laidlawii, four primers were prepared based on the 23S rRNA regions. Using these primers and a minimum of 100 fg of mycoplasma genomic DNA, the 23S rRNA regions of these eight mycoplasma species were consistently amplified by real-time PCR. In contrast, no specific specific amplification product was observed using DNA templates prepared from various mammalian cell lines. Frozen and cultured samples of several cell lines were tested for mycoplasma contamination to evaluated the utility of this method. Of 25 samples that tested positive for mycoplasma by Hoechst staining, which requires two passages of cell cultures started from frozen samples, mycoplasma was detected by real-time PCR in 24 samples of cell extracts prepared directly from frozen samples. When cultured samples were used for this assay, the accuracy of the diagnoses was further improved. Thus, this technique, which is simple, rapid, and sensitive enough for practical application, in suitable for handling many samples and for routine screening for mycoplasma contamination of cell cultures.     

Cell surface gamma-glutamyl transpeptidase in live cultures.

A physiological assay for measuring surface accessible gamma-glutamyl transpeptidase activity in adherent, living cultures is described. Cell surface transpeptidase activity remained linear throughout a 60-min time course over a wide range of cell densities. In addition, the assay conditions have neither acute nor long-term effects on cell growth potential, cellular morphology, or cell surface transpeptidase activity levels. As a result, cell surface transpeptidase activity can be continually evaluated in the same cultures during proliferation. The assay appears to be specific for cell surface transpeptidase and can be used to study the partitioning of the enzyme between substrate-accessible and substrate-inaccessible pools. This method utilizes an automated microtiter plate reader for the spectrophotometric quantification of small aliquots removed from cultures incubated with the chromogenic substrate L-gamma-glutamyl-p-nitroanilide. The use of a microtiter plate autoreader and the minimal handling of the cells permit a large number of cultures to be assayed with a substantial reduction in the time required to measure surface transpeptidase activity. The assay described is a nondestructive means for studying cell surface-accessible gamma-glutamyl transpeptidase catalytic activity within the microenvironment of the living culture.     

Comprehensive profiling of radiosensitive human cell lines with DNA damage response assays identifies the neutral comet assay as a potential surrogate for clonogenic survival

In an effort to explore the possible causes of human radiosensitivity and identify more rapid assays for cellular radiosensitivity, we interrogated a set of assays that evaluate cellular functions involved in recognition and repair of DNA double-strand breaks: (1) neutral comet assay, (2) radiation-induced γ-H2AX focus formation, (3) the temporal kinetics of structural maintenance of chromosomes 1 phosphorylation, (4) intra-S-phase checkpoint integrity, and (5) mitochondrial respiration. We characterized a unique panel of 19 "radiosensitive" human lymphoblastoid cell lines from individuals with undiagnosed diseases suggestive of a DNA repair disorder. Radiosensitivity was defined by reduced cellular survival using a clonogenic survival assay. Each assay identified cell lines with defects in DNA damage response functions. The highest concordance rate observed, 89% (17/19), was between an abnormal neutral comet assay and reduced survival by the colony survival assay. Our data also suggested that the neutral comet assay would be a more rapid surrogate for analyzing DNA repair/processing disorders.     

Determination of cell number in monolayer cultures

Determining the cytostatic or cytotoxic effects of various conditions on monolayer cells requires techniques that are rapid, reproducible, and able to monitor these effects as a function of time. Methods currently used to monitor cytostasis or cytotoxicity are either static or indirect; that is, they are designed to test effects of various treatments either at single time points or on associated cellular processes, such as membrane integrity. Because of these limitations in extant techniques, we undertook this study to improve methods for the rapid determination of cell number in monolayer cultures. We have arrived at conditions of staining cell nuclei with crystal violet under fixed regimens which allow rapid and reproducible quantification of cell number in cultures grown in 24-well miniwells. Quantification is possible by solubilizing the adsorbed dye into a solution of Triton X-100 and determining optical density (O.D.) using spectrophotometry. The present communication documents that O.D. is linearly related to cell number with a sensitivity of ca. 500 cells and that the technique is applicable to study agents which affect cell proliferation.     

A Procedure for the Measurement of Hormone Receptors Using a Cell Harvesting Machine

Abstract

A procedure has been developed that will rapidly measure the binding of hormones and growth factors to suspensions of intact cells. Radiolabeled ligand is bound to cells in 96-well cluster plates, and the cells are then collected and washed on glass fiber filters using a cell harvester. Up to twelve wells can be processed simultaneously in less than a minute. This rapid washing and harvesting method is convenient to use with a large number of samples, and the amount of bound ligand is saturable and proportional to cell number. This procedure should be applicable to suspension cultures, loosely adhering monolayer cultures, and other cellular preparations that will bind to porous filters. Using this procedure, we have shown that HeLa S₃ cells grown in suspension cultures saturably bind to epidermal growth factor (EGF). The quantity of EGF bound to HeLa S₃ cells can be increased by adding glucocorticoids to the culture medium.     

A high-throughput method to measure the sensitivity of yeast cells to genotoxic agents in liquid cultures

The sensitivity of yeast Saccharomyces cerevisiae to DNA damaging agents is better represented when cells are grown in liquid media than on solid plates. However, systematic assessment of several strains that are grown in different conditions is a cumbersome undertaking. We report an assay to determine cell growth based on automatic measurements of optical densities of very small (100 microl) liquid cell cultures. Furthermore, an algorithm was elaborated to analyze large data files obtained from the cell growth curves, which are described by the growth rate--that starts at zero and accelerates to the maximal rate (mu(m))--and by the lag time (lambda). Cell dilution spot test for colony formation on solid media and the growth curve assay were used in parallel to analyze the phenotypes of cells after treatments with three different classes of DNA damaging agents (methyl methanesulfonate, bleomycin, and ultraviolet light). In these experiments the survival of the WT (wild type) and a number of DNA repair-deficient strains were compared. The results show that only the cell growth curve assay could uncover subtle phenotypes when WT cells, or mutant strains that are only weakly affected in DNA repair proficiency, were treated with low doses of cytotoxic compounds. The growth curve assay was also applied to establish whether histone acetyltransferases and deacetylases affect the resistance of yeast cells to UV irradiation. Out of 20 strains tested the sir2delta and rpd3delta cells were found to be more resistant than the WT, while gcn5delta and spt10delta cells were found to be more sensitive. This new protocol is sensitive, provides quantifiable data, offers increased screening capability and speed compared to the colony formation test.     

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.     

Cryopreservation of Cortical Tissue Blocks for the Generation of Highly Enriched Neuronal Cultures

In this study, we outline a standardized protocol for the successful cryopreservation and thawing of cortical brain tissue blocks to generate highly enriched neuronal cultures. For this protocol the freezing medium used is 10% dimethyl sulfoxide (DMSO) diluted in Hank''s Buffered Salt Solution (HBSS). Blocks of cortical tissue are transferred to cryovials containing the freezing medium and slowly frozen at -1°C/min in a rate-controlled freezing container. Post-thaw processing and dissociation of frozen tissue blocks consistently produced neuronal-enriched cultures which exhibited rapid neuritic growth during the first 5 days in culture and significant expansion of the neuronal network within 10 days. Immunocytochemical staining with the astrocytic marker glial fibrillary acidic protein (GFAP) and the neuronal marker beta-tubulin class III, revealed high numbers of neurons and astrocytes in the cultures. Generation of neural precursor cell cultures after tissue block dissociation resulted in rapidly expanding neurospheres, which produced large numbers of neurons and astrocytes under differentiating conditions. This simple cryopreservation protocol allows for the rapid, efficient, and inexpensive preservation of cortical brain tissue blocks, which grants increased flexibility for later generation of neuronal, astrocyte, and neuronal precursor cell cultures.     

The determination of cellular viability of hybridoma cells in microtitre plates: A colorimetric assay based on neutral red

A colorimetric assay utilising Neutral Red (C.I. 50040), a nuclear stain, was developed to determine the cellular viability of hybridoma cells in microtitre plates. A linear correlation (r=0.99) was found to exist between the uptake of Neutral Red by viable cells and the viable cell count determined by Trypan blue exclusion test. The linearity stretched over the range of cell concentrations normal in batch cultures (2–30×10⁴/0.2 ml) with as little as ±6% intra-plate well-to-well variation and ±10.2% inter-assay variation.Microscopical examinations of viable hybridoma cells stained with Neutral Red showed that it was located in the nucleus. The possble bifunctional activity of the Neutral Red assay as a test for cellular viability and estimating the DNA content of hybridoma cells is discussed along with its application in a drug screening programme.     

Rapid determination of DNA synthesis in adherent cells grown in microtiter plates

A specific, rapid, and economical method for measuring the extent of DNA synthesis in adherent rat hepatoma H4-II-E cells grown in 96-well microtiter plates is described. The adherent cells were pulsed for 1 h with [methyl-3H]thymidine, released from the substratum by trypsinization, and collected on fiberglass filters with a MASH II cell harvester. The amount of radioactivity incorporated was directly proportional to the number of cells per well. Growth curves generated by measuring [methyl-3H]thymidine incorporation and counting the number of cells per well were identical. Experiments with inhibitors of DNA, protein, and RNA synthesis demonstrated that this method selectively measured DNA synthesis. In addition, [3H]thymidine uptake showed excellent correlation with autoradiographic assessment of DNA synthesis. This specific and sensitive method for determining DNA synthesis in microtiter cultures should facilitate studies of effects of various growth-controlling agents on epithelial, fibroblastic, and other cells which grow as adherent cells in culture.     

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.     

A simplified polyethylenimine-mediated transfection process for large-scale and high-throughput applications

Transient gene expression in mammalian cells is a valuable alternative to stable cell lines for the rapid production of large amounts of recombinant proteins. While the establishment of stable cell lines takes 2-6 months, milligram amounts of protein can be obtained within a week following transfection. The polycation polyethylenimine (PEI) is one of the most utilized reagents for small- to large-scale transfections as it is simple to use and, when combined with optimized expression vectors and cell lines, provides high transfection efficiency and titers. As with most transfection reagents, PEI-mediated transfection involves the formation of nanoparticles (polyplexes) which are obtained by its mixing with plasmid DNA. A short incubation period that allows polyplexes to reach their optimal size is performed prior to their addition to the culture. As the quality of polyplexes directly impacts transfection efficiency and productivity, their formation complicates scalability and automation of the process, especially when performed in large-scale bioreactors or small-scale high-throughput formats. To avoid variations in transfection efficiency and productivity that arise from polyplexes formation step, we have optimized the conditions for their creation directly in the culture by the consecutive addition of DNA and PEI. This simplified approach is directly transferable from suspension cultures grown in 6-well plates to shaker flasks and 5-L WAVE bioreactors. As it minimizes the number of steps and does not require an incubation period for polyplex formation, it is also suitable for automation using static cultures in 96-well plates. This "direct" transfection method thus provides a robust platform for both high-throughput expression and large-scale production of recombinant proteins.     

Effects of growth phase,diel cycle and macronutrient stress on the quantification of Heterosigma akashiwo using qPCR and SHA

The development of molecular probe technologies over the last several decades has enabled more rapid and specific identification and enumeration of phytoplankton species compared to traditional technologies, such as light microscopy. Direct comparisons of these methods with respect to physiological status, however, are sparse. Here we directly compare quantitative real-time PCR (qPCR) and sandwich hybridization assay (SHA) for enumerating the raphidophyte Heterosigma akashiwo at several points during its growth phase, over a diel cycle and with macronutrient stress in laboratory cultures. To ensure consistency between comparisons, a single cellular homogenate was generated from each culture and split for analysis by qPCR and SHA. Since the homogenate was generated from the same number of cells during each experiment, results reflect changes in nucleic acid content (rRNA and DNA) at each time point or in response to environmental conditions relative to a reference sample. Results show a greater level of precision in SHA results which contributed to significant (2–3 fold) differences in rRNA content per cell in several of these analyses. There was significantly greater rRNA content during lag and exponential phases compared to stationary phase cultures, and a significant decrease in rRNA content during the light cycle compared to cells harvested in the dark. In contrast, there were no significant differences in DNA content per cell as determined by qPCR over a diel cycle or during different growth phases. There was also no decrease in either rRNA or DNA content for cultures under low P conditions compared to nutrient replete conditions. However, both rRNA and DNA content were significantly lower under N stress when compared to nutrient replete conditions. Results of this study suggest that growth stage, nutrient stress and cell cycle may impact molecular analyses, and that physiological status should be taken into account when using these methods for HAB monitoring.     

Identification of Major Factors Influencing ELISpot-Based Monitoring of Cellular Responses to Antigens from Mycobacterium tuberculosis

A number of different interferon-γ ELISpot protocols are in use in laboratories studying antigen-specific immune responses. It is therefore unclear how results from different assays compare, and what factors most significantly influence assay outcome. One such difference is that some laboratories use a short in vitro stimulation period of cells before they are transferred to the ELISpot plate; this is commonly done in the case of frozen cells, in order to enhance assay sensitivity. Other differences that may be significant include antibody coating of plates, the use of media with or without serum, the serum source and the number of cells added to the wells. The aim of this paper was to identify which components of the different ELISpot protocols influenced assay sensitivity and inter-laboratory variation. Four laboratories provided protocols for quantifying numbers of interferon-γ spot forming cells in human peripheral blood mononuclear cells stimulated with Mycobacterium tuberculosis derived antigens. The differences in the protocols were compared directly. We found that several sources of variation in assay protocols can be eliminated, for example by avoiding serum supplementation and using AIM-V serum free medium. In addition, the number of cells added to ELISpot wells should also be standardised. Importantly, delays in peripheral blood mononuclear cell processing before stimulation had a marked effect on the number of detectable spot forming cells; processing delay thus should be minimised as well as standardised. Finally, a pre-stimulation culture period improved the sensitivity of the assay, however this effect may be both antigen and donor dependent. In conclusion, small differences in ELISpot protocols in routine use can affect the results obtained and care should be given to conditions selected for use in a given study. A pre-stimulation step may improve the sensitivity of the assay, particularly when cells have been previously frozen.     

Microtiter plate radioreceptor assay for tumor necrosis factor and its receptors in large numbers of samples.

We developed a rapid radioreceptor assay for tumor necrosis factor (TNF) and its receptors by utilizing serum-coated 96-well plates. This assay has an advantage over the traditional tube assay in speed, number of simultaneous samples, and amount of material needed for the assay. As many as 200 samples were assayed in less than 3 h. The usefulness of this assay to determine TNF and its cell surface receptors and to screen for TNF inhibitors in large numbers of biological fluids from patients is demonstrated. This assay may also prove useful for determining the activity of competitive antagonists of TNF.     

Fragmented DNA and apoptotic bodies document the programmed way of cell death in hybridoma cultures

Markers of apoptosis were followed in batch hybridoma cultures carried out in protein-free medium. Samples were collected on day 0, representing early exponential phase (viability 91%), and on day 8, corresponding to late stationary phase (viability 8%). The apoptotic index reflecting the relative number of bodies insoluble in 6 M guanidinium hydrochloride in the culture of day 8 (30%) exceeded markedly the index in the culture of day 0 (2.5%). A gel chromatography on Sepharose 2B was developed for quantitative evaluation of fragmented cellular DNA. This analysis, including a correction for nonspecific fragmentation, showed that on day 8 more than 30% of cellular DNA was fragmented, whereas on day 0 it was less than 5%. Control necrotic cells prepared by rapid killing in 1% sodium azide displayed a low apoptotic index (2.4%) and low DNA fragmentation. Electrophoretic patterns in agarose gel showed a typical “ladder” of fragments in the DNA sample of day 8. The demonstration of fragmented cellular DNA and of the high incidence of apoptotic bodies at late stationary phase adds substantial weight to the view that in hybridoma cultures apoptosis represents the prevalent mode of cell death.     

Modification of the phosphoketolase assay for rapid identification of bifidobacteria

The phosphoketolase assay is commonly used as a definitive criterion for identification of bifidobacteria. A limitation of the assay is the time-consuming process of cell disruption, either by use of the French Pressure Cell or by sonication. We have replaced the time consuming cell disruption process with a more rapid cell membrane disruption process by pretreating cells with the detergent hexadecyltrimethylammonium bromide (cetrimonium bromide, CTAB). The effect of no pretreatment, sonication or the addition of CTAB (0.45 mg/ml) on color development in the phosphoketolase assay was tested using pure cultures of bifidobacteria and lactobacilli. No phosphoketolase activity was observed with bifidobacterial cultures without cell disruption or with lactobicilli that had undergone cell disruption. All bifidobacterial cultures gave a similar color formation whether sonication or CTAB addition was used to disrupt cells. Use of CTAB to disrupt cell membranes is an effective alternative to the time consuming traditional cell disruption procedures and increases the number of cultures that can be simultaneously assayed and presumptively identified using the phosphoketolase assay.     

Spontaneous revertants in modified S. typhimurium mutagenicity tests employing elevated numbers of the tester strain

It has been proposed that increases in the number of bacteria applied to each plate can enhance the sensitivity of the Ames S. typhimurium mutagenicity assay. These procedures have the potential to elevate the number of spontaneous revertants (SR) by increasing the contribution of pre-existing revertants (PER) present before application of the bacteria to the limited histidine test plates. We have investigated the contribution of PER when 10(9) bacteria are applied to the plates and found that the number of PER is dependent on the handling and storage of the cultures used to inoculate the overnight broth. The average number of PER/10(9) viable bacteria after overnight growth in broths inoculated from a frozen permanent, lyophilized permanent, master plate, and an isolated colony, of TA98 were 267, 188, 57 and 13 respectively. The resultant elevation of the number of SR for a strain may result in a failure to identify a mutagenic response. It is recommended that the number of PER be monitored in any modification of the Ames test that makes use of elevated numbers of bacteria.     

A rapid assay for fusion of embryonic chick myoblasts

A rapid and sensitive assay for measuring myoblast fusion in suspension cultures of embryonic chick pectoral myoblasts is described. Fusion-competent cells are generated by growth in suspension using a low calcium medium. Fusion-promoting levels of calcium are added, and the suspensions incubated for 1–6 h. The cells are then trypsinized to disperse cellular aggregates and sized in a Coulter particle counter. This assay minimizes many of the artifacts inherent in measurements of fusion in monolayer cultures, and is designed for the rapid screening of agents for their effects on fusion.