Search

Measurement of proteasome activity is fast becoming a commonly used assay in many laboratories. The most common method to measure proteasome activity involves measuring the release of fluorescent tags from peptide substrates in black microplates. Comparisons of black plates used for measuring fluorescence with different properties show that the microplate properties significantly affect the measured activities of the proteasome. The microplate that gave the highest reading of trypsin-like activity of the purified 20S proteasome gave the lowest reading of chymotrypsin-like activity of the 20S proteasome. Plates with medium binding surfaces from two different companies showed an approximately 2-fold difference in caspase-like activity for purified 20S proteasomes. Even standard curves generated using free 7-amino-4-methylcoumarin (AMC) were affected by the microplate used. As such, significantly different proteasome activities, as measured in nmol AMC released/mg/min, were obtained for purified 20S proteasomes as well as crude heart and liver samples when using different microplates. The naturally occurring molecule betulinic acid activated the chymotrypsin-like proteasome activity in three different plates but did not affect the proteasome activity in the nonbinding surface microplate. These findings suggest that the type of proteasome activity being measured and sample type are important when selecting a microplate.

Abstract We present a real-time, high-throughput, and cost-effective method of detecting apoptosis in vitro using a previously developed reagent that detects caspase activation by fluorescence. Current methods of assessing apoptosis fail to account for the dimension of time, and thus are limited in data yielded per sample. This reagent allows real-time detection of apoptosis, but until now has been restricted to a costly automated detection system. Here, we describe apoptosis detection with the Essen Bioscience IncuCyte® Caspase-3/7 Reagent using a multimode microplate reader, a common instrument in biological laboratories, which may be used prior to or in lieu of the automated system. This modified microplate reader apoptosis assay was validated against the established automated system, and was shown to detect a strong dose-response relationship (automated system r2 = 0.9968, microplate reader r2 = 0.9924). We also propose a quick and reliable method of quantifying cell density by Hoechst 33342 nuclear staining in microplates (r2 = 0.8812 between Hoechst signal and cell density). We assert that the dimension of time should not be overlooked, and that the method presented here is an accessible strategy for many researchers due to low startup cost and precise detection of apoptosis in real time. Highlights • Popular methods of apoptosis detection yield a single reading per sample • Accounting for kinetics of the apoptotic response provides more robust and precise data outputs • Current real time methods of apoptosis detection are either expensive or have low sample capacity • A high-throughput microplate reader based assay is shown here to accurately detect apoptosis in real time [ABSTRACT FROM AUTHOR]

MicroRNAs (miRNAs) have key roles in gene expression and can be employed as biomarkers for early diagnosis of various diseases, especially cancers. Detection of miRNAs remains challenging and often requires detection platforms. Here, a horseradish peroxidase (HRP)-assisted hybridization chain reaction (HCR) for colorimetric detection of miR-155 was described. In the presence of target miRNA, the capture probe immobilized on the microplate sandwiched the target miR-155 with the 3′ end of the reporter probe. Another exposed part of the RP at the 5'end triggered HCR producing double-stranded DNA polymers with multiple fluorescein isothiocyanates (FITC) for signal amplification. Finally, multiple HRP molecules were immobilized onto the long-range DNA nanostructures through FITC/anti-FITC monoclonal antibody interactions on the microplate for visualization by tetramethylbenzidine/H 2 O 2 system and the colorless substrate turned into the blue product. To obtain accurate data, the absorbance at 450 nm was calculated by microplate reader. The detection limit was 31.8 fM (3.18 amol). Furthermore, this biosensor showed high specificity and was able to discriminate sharply between target miRNA and mismatched sequences. And this approach could be easily applied to the detection of miR-155 in serum sample, thereby ascribing it for a wide application. [ABSTRACT FROM AUTHOR]

Traditional assays for secreted proteins include methods such as Western blot and enzyme-linked immunosorbent assay (ELISA) detection of the protein in the cell culture medium. We describe a method for the detection of a secreted protein based on fluorescent measurement of an mCherry fusion reporter. This microplate reader-based mCherry fluorescence detection method has a wide dynamic range of 4.5 orders of magnitude and a sensitivity that allows detection of 1 to 2 fmol fusion protein. Comparison with the Western blot detection method indicated greater linearity, wider dynamic range, and a similar lower detection threshold for the microplate-based fluorescent detection assay of secreted fusion proteins. An mCherry fusion protein of matrix metalloproteinase-9 (MMP-9), a secreted glycoprotein, was created and expressed by transfection of human embryonic kidney (HEK) 293 cells. The cell culture medium was assayed for the presence of the fluorescent signal up to 32 h after transfection. The secreted MMP-9–mCherry fusion protein was detected 6 h after transfection with a linear increase in signal intensity over time. Treatment with chloroquine, a drug known to inhibit the secretion of many proteins, abolished the MMP-9–mCherry secretion, demonstrating the utility of this method in a biological experiment. [ABSTRACT FROM AUTHOR]

Abstract: Microplates for use in resource-limited laboratories should ideally not require processes that involve substantial large-scale production in order to be viable. We describe and demonstrate here an approach of using a silicone sheet with holes, conveniently cut out precisely using an inexpensive cutting plotter to correspond with regions where liquid is to be dispensed, and attaching it to a transparency to create very thin well arrays. With this, the contact angle hysteresis behavior of liquid could be harnessed to produce taller drop shapes so that the fiber probe used could read in the emitted light more effectively. Experimentation conducted revealed fluorescence measurements that were significantly more sensitive than standard microplates, notwithstanding that smaller volumes of liquid were needed. This was achieved using both the fiber optic and imaging evaluation modes. The two methods investigated, one with a lid placed and one without, showed the latter to produce marginally more sensitive readings as opposed to improved immunity from the environment with the former. These favorable measurement characteristics were found to be achievable with an estimated production cost of AU $0.40 and fabrication times of 3.5min (96 wells) and 6.5min (384 wells) per plate. [Copyright &y& Elsevier]

Abstract: Using both experimental assays and fluid-dynamic finite element simulation models, we directly compared the achievable performance limits of four distinct assay configurations for label-free detection of an analyte from a test sample on a biosensor surface. The assay configurations studied in this work included a biosensor incorporated into the bottom surface of a microplate well and a microfluidic channel. For each configuration, we compared assay performance for the scenario in which the entire bottom surface of the fluid-handling vessel is coated with capture ligands with assay performance for the scenario in which the capture ligands are applied in the form of localized spots. As a model system, we used detection of the protein biomarker tumor necrosis factor-alpha (TNF-α) using immobilized TNF-α capture antibody. Results show that the microfluidic assay format dramatically reduces the time required to establish a stable equilibrium. Spot-based assays are advantageous for microplate-based detection for reducing the time required for equilibrium sensor response. The results derived are generally applicable to any label-free biosensor technology and any ligand–analyte system with adjustable variables that include sensor mass density sensitivity, analyte–ligand adsorption/desorption rate constants, immobilized ligand density, flow channel geometry, flow rate, and spot size. [Copyright &y& Elsevier]

Abstract: Among many colorimetric methods for carbohydrate analysis, the phenol–sulfuric acid method is the easiest and most reliable method. It has been used for measuring neutral sugars in oligosaccharides, proteoglycans, glycoproteins, and glycolipids. This method is used widely because of its sensitivity and simplicity. In its original form, it required 50–450nmol of monosaccharides or equivalent for analysis and thus is inadequate for precious samples. A scaled-down version requiring only 10–80nmol of sugars was reported previously. We have now modified and optimized this method to use 96-well microplates for high throughput, to gain greater sensitivity, and to economize the reagents. This modified and optimized method allows longer linear range (1–150nmol for Man) and excellent sensitivity. Moreover, our method is more convenient, requiring neither shaking nor covering, and takes less than 15min to complete. The speed and simplicity of this method would make it most suitable for analyses of large numbers of samples such as chromatographic fractions. [Copyright &y& Elsevier]

A fluorescence-based microplate assay was developed to quantify cell death based upon the measurement of glucose-6-phosphate dehydrogenase (G6PD) activity. G6PD is a cytosolic enzyme and leaks from cells when plasma membrane integrity is compromised. In this assay, cell death is measured by correlating the activity of extracellular G6PD to the reduction of resazurin to the fluorescent product, resorufin, via a coupled-enzyme reaction. The coupled-enzyme reaction permits rapid signal amplification from small amounts of G6PD, an advantage over assays based on resazurin alone. This assay is rapid, nontoxic, and amenable to high-throughput screening. The assay has a Z′ factor of 0.78. [Copyright &y& Elsevier]

Monocyte chemoattractant protein-1 (MCP-1) is a potential therapeutic target for the treatment of several inflammatory conditions, including rheumatoid arthritis and chronic obstructive pulmonary disease. Current cell-based assays for MCP-1 use monocyte chemotaxis or calcium flux as a readout. Here, we describe an alternative bioassay based on MCP-1-induced phosphorylation of the mitogen-activated protein kinases (MAPK) p44 (ERK1) and p42 (ERK2). Adherent cells expressing the MCP-1 receptor CCR2B are treated with MCP-1 in 96-well plates in the presence or absence of inhibitors, fixed and permeabilized with methanol, and then probed with a monoclonal antibody that selectively recognizes the doubly phosphorylated form of p44/42 MAPK. Bound antibody is detected with a secondary antibody-peroxidase conjugate and a chromogenic substrate. The phosphorylation of p44/42 MAPK as detected in this assay peaks after 3–5 min of MCP-1 treatment, and the concentration of MCP-1 required for half-maximal p44/42 MAPK phosphorylation is 1–3 nM. MCP-1-induced phosphorylation of p44/42 MAPK is dependent upon the expression of CCR2B. The assay can be used for screening and characterization of small molecule inhibitors and antibodies blocking the binding of MCP-1 to its receptor. Since the assay is rapid and simple, it may represent a useful alternative to chemotaxis or calcium mobilization assays for the analysis of MCP-1 inhibitors. [Copyright &y& Elsevier]

We have successfully configured a new ultrasensitive fluorescent phosphate assay that detects free phosphate in solution through the formation of the fluorescent product resorufin. The phosphate assay relies on coupling phosphate generation to purine nucleoside phosphorylase, xanthine oxidase, and horseradish peroxidase. The response is excellent in the nanomolar range, being linear between 50 nM and 5 μM phosphate. This method is more sensitive (more than 10-fold) than other reported methods and is amenable to miniaturization. In particular, we have demonstrated the utility of this new method in a format suitable for ultra-high-throughput screening. [Copyright &y& Elsevier]

Abstract: The immense cost effectiveness of using transparencies as analyte handling implements in microplate instrumentation offers the possibility of application even in resource-limited laboratories. In this work, a standard microplate was adapted to serve as the permanent base for disposable scribed transparencies. The approach is shown to ameliorate evaporation, which can affect assay accuracy when analytes need to be incubated for some time. It also offers assurance against fluorescence measurement errors due to the cross-talk of samples from adjacent wells. [Copyright &y& Elsevier]

Abstract: Endo-polygalacturonase (endo-PGase) activity determinations generally rely on viscosity changes or reducing sugar ends produced by this activity over polygalacturonic acid. Torres and coworkers [Enzyme Microb. Technol. 48 (2011) 123–128] showed that ruthenium red (RR) is useful for endo-PGase determination. In this article, we present a high-throughput liquid-based endo-PGase assay based on the RR method and compare it with the viscosity determination method. The reduced assay uses a small volume of enzyme solution, 40μg of polygalacturonic acid, and 45μg of RR for each sample determination. Furthermore, we obtained an interconversion factor for RR and viscosity activities. [Copyright &y& Elsevier]

Abstract: As erythrocyte-derived extracellular adenosine triphosphate (ATP) gains recognition as a key vasodilator, its accurate determination is critical. Erythrocytes’ high hemoglobin content can act as an inner filter when measuring ATP concentrations by chemiluminescence. We evaluated two approaches to correct for this matrix effect: addition of cell-free hemoglobin to the ATP standards and standard addition of ATP to erythrocyte suspensions. In addition, we reduced sample hematocrit to minimize the absorbance. We conclude that extracellular ATP should be determined in erythrocyte suspensions at 0.06 to 0.004% hematocrit. This gives robust signals without matrix effects and requires only microliters of blood. [Copyright &y& Elsevier]

Abstract: Two important challenges in microplate instrumentation are to achieve full well sample coverage and complete mixing. An effective approach of using superhydrophobic rods to accomplish these challenges is reported here. Experiments conducted showed that analytes above 50μl could be made to completely cover the bottom of 96-well standard and transparency microplates. Complete mixing was accomplished by moving the rod parallel to the well bottom while contacting the liquid. The approach is simple and controlled, and it minimizes the problems of spillage and cross-contamination. It works with analytes with varied volumes and of different viscosities present in each well of the microplate. [Copyright &y& Elsevier]

Abstract: Smaller fluid samples can offer higher sensitivity under analysis and allow more samples from a sparse specimen. However, for a microplate well there is a minimum volume requirement arising from the need for the fluid/air interface to make contact only on the walls of the well, a condition which allows high-quality undistorted optical imaging due to the minimum curvature of this interface. In this work, mechanical vibration is investigated as a method to significantly decrease this minimum volume requirement. Furthermore, low-frequency excitation is shown to be ideal for handling multiple wells (such as in a microplate) simultaneously. [Copyright &y& Elsevier]