featuring the Echo Acoustic Technology
TITLES and AUTHORS
The serial dilution method is standard practice in the preparation of dose-response series for IC50 determination. However, it is well recognised that inadequacies in the liquid handling or mixing technique will affect the dilution ratio and hence the compound concentration and any errors will be compounded during each successive serial dilution, mix and transfer. A recent poll of end users ranked better precision, particularly at lower drug concentrations, and the reduction in compound precipitation as the improvements in dose-response analysis they most desired. In addition, it is now suspected that hydrophobic compounds may be lost from solution during aqueous serial dilutions and absorbed to intermediate plastic surfaces. This in turn adds to concern over the reliability of the results generated and the extent to which they are a true reflection of the potency of the compounds being evaluated. As part of the general drive to enhance the quality of screening data generated researchers are investigating strategies based on the direct dilution of micro-volumes of compound (i.e. on a volumetric basis). These investigations have been aided by the availability of low volume dispensing systems with good precision at low nL dispense volumes and a relatively wide dynamic range. Some groups are now reporting that IC50 values of compounds tend to be lower (more active) when the concentrations are made via direct dilutions. It is increasingly evident that direct dilution has a future role to play in dose-response analysis and where acoustic droplet ejection is preferentially deployed additional benefits will be derived in terms of reduced waste stream generated, less source material used and no cross-contamination.
Acoustic-based non contact droplet ejection compound transfer methods have become more widely utilized due to advantages demonstrated over traditional methods. These advantages include minimizing compound usage and subsequent conservation of compound bank as well as improved data quality through removal of serial dilution cross-contamination issues, reduction in false negative rates, lower consumable costs through reduced plastic and reagent wastage and lower final assay DMSO concentration for poorly tolerant assays.
Within the Lead Identification arena we have utilized acoustic based compound transfer where a requirement for flexible operation is appropriate software to enable easy design of transfer protocols. We have evaluated and utilized the Labcyte Echo™ Dose Response version 1.1.0 software for a range of direct microtiter to microtitre plate applications on the nanolitre transfer technology of the Labcyte® Echo™ 555 liquid handler.
The Echo™ dose-response software enables the user to define the layout of the source and destination plates, specify the number of points in the dose response curve, and transfer various concentrations and volumes of compounds to the destination plates. The software runs the liquid transfer as well as the DMSO back-fill, and generates a report of the survey and transfer results.
Acoustic droplet ejection has already been shown to be successful for the transfer of liquids including DMSO, mineral oil, small chain alcohols and acetonitrile. In addition to this, the transfer of aqueous based phosphate buffered saline containing protein (bovine serum albumin) has also been reported. We present data showing that human A549 cells that produce constitutively active Renilla luciferase can also be transferred using this technology. Quantitative evaluation of the extent of transfer of cells was made by the determination of cell viability and measurement of the emission of light subsequent to detergent based cell lysis and addition of Renilla luciferase substrate. Only intact cells that were transferred would be expected to be viable and express Renilla luciferase protein.
Since the commercial introduction of microarray technology for differential gene expression analysis in the late 1990s, array usage has expanded into a broad range of applications. Acoustic droplet ejection (ADE) is a new and unique technology that enables fast fabrication of arrays from nanoliter and picoliter droplets. ADE uses acoustic energy to transfer ultra-low volumes of many different biological reagents. There are no nozzles or pin tools, thereby eliminating washing steps, cross-contamination and clogging. We demonstrate four array applications using two different ADE instrument platforms. 1) We create multiplex slide arrays by transferring 2.5 nL droplets of protein solution from 384- and 1536-well plates onto glass microscope slides using arraying software developed in house for the Echo® series liquid handlers. 2) We create arrays in the bottom of 96-well microplates by transferring 2.5 nL droplets of glycerol-based solutions onto the well bottoms. 3) We demonstrate filling microstructure arrays and spot-on-spot reagent deposition using the Portrait™ reagent multi-spotter to transfer 170 pL droplets of fluorescent dye onto pre-existing spot locations. 4) We demonstrate making arrays of reagents on tissue sections for in situ biochemical reactions by transferring 170 pL droplets of MALDI matrix onto tissue sections for MALDI imaging mass spectrometry. ADE is a precise, flexible and fast solution for a wide range of arraying applications.
G protein-coupled receptors (GPCRs) are involved in various physiological processes such as the regulations of behavior, mood and immune system activity. GPCRs are popular targets in drug discovery,
and a well-designed assay can speed up the discovery of novel drug candidates. The Promega cAMP-Glo™ Assay monitors cAMP production in cells in response to the effect of an agonist or test compound on GPCRs. The assay is based on the principle that cyclic AMP (cAMP) stimulates protein kinase A (PKA) holoenzyme activity, decreasing available ATP and leading to decreased light production in a luciferase reaction. Together with the Labcyte® Echo® 555 acoustic liquid handler and the Deerac Fluidics™ Equator™ HTS reagent dispenser, compounds can be screened in 1536-well format for effects on GPCRs. Implementing a high-throughput miniaturized GPCR assay as demonstrated in this poster allows cost-effective screening to identify lead modulators of GPCR signaling.
Current methods of optical high content screening (HCS) involve cells cultured in Petri dishes, 96, 384 and 1536 well plates and on microscope slides. The costs of running many experiments in microtiter plates is high due to the amount of cell culture media, the number of cells required, and the amount of reagent used. We describe herein a high-density cell containment HCS device termed cellTRAY™ and the loading of live cells employing acoustic droplet ejection (ADE) technology. cellTRAY enables the retention of multiple cells in an ordered microarray in an optically clear glass substrate. The cell array enables automated processing, simultaneous monitoring and image analysis of a large matrix of cells. The cellTRAY includes 8 separate sub-regions and comes in two well sizes. With the 300 μm well size there are a total of 640 microwells contained on a 1” x 3” glass substrate, the size of a standard microscope slide. The cellTRAY also incorporates microfluidic channels that enable the precise delivery of liquids such as cell culture media or reagents. Traditionally cells are loaded into microplates using pipette based dispensing technologies; however, recently developed automated, non-contact, nozzlefree acoustic droplet ejection (ADE) technology offers significant advantages for the transfer of live cells. We describe the ADE loading of live cells in 200 pL droplets into the sub-microliter wells of the cellTRAY cell containment device and subsequent optical image analysis of the cells. The combination of ADE and cellTRAY provides a powerful new enabling platform for performing high content cell-based analysis and screening assays for new target and drug discovery applications.
Objective: Assess performance of acoustic droplet ejection and whole-well fluorescence imaging for quantitative high-throughput cellular dispensing and measurement. Methods: Source plates contained CMFDA (Invitrogen)-labeled CHO cells resuspended in PBS from 1- to 2x106 cells/mL. Cells were dispensed over a range of 0-400 droplets of 2.5 nL each (i.e., final dispensed volumes of 0-1000 nL) using acoustic droplet ejection (ADE; Echo® 555 liquid handler, Labcyte Inc.). The cell concentration curve was dispensed in both forward and reverse direction across the receiving plate to determine whether cells settled in the source wells over the dispensing time. The receiving 384-well plates contained 40 μL/well growth medium + 10% FCS and after dispensing, the plates were incubated at 37°C/5% CO2. Whole-well images of the 384-well plate were acquired using a 488 nm laser scanning platform (IsoCyte™, Blueshift Biotechnologies). Total cell counts per well was determined using the IsoCyte software. A solution of saponin (Sigma Aldrich) and propidium iodide (Invitrogen) was added at 40 μL/well and the plates were scanned again to determine nuclear counts. Results: These tests demonstrated that the cell counts per well increased linearly with increasing dispense volume with both CMFDA and PI stains. A starting concentration of 1x106 cells/mL yielded about one cell per 2.5 nL droplet across all dispense volumes, with the expected doubling at 2x106 cells/mL. Among 24 replicates for each dispense volume above 5 nL, the average CV for the cell counts was 19%.
Conclusions: The Echo ADE liquid handling system can be used to dispense live cells into high-density microplates in a quantitative manner. Total cell counts in each well can be rapidly determined using fluorescent dyes and the IsoCyte laser scanning platform. Some applications for high-throughput low-cell-number dispensing and fluorescent quantitation include cell health measurements, cell cloning, and RNAi studies.
This review assesses the quality of the data acquired over a 13-week period from a High-Content Analysis screening project that used 297 unique cell lines. This article also evaluates the proficiency of a “tipless” (i.e., does not use disposable tips) full-automation design used for this project that prioritizes intralab system mobility and system configuration mutability. The request to assay a large number of cell lines with poorly characterized growth rates led us to devise an MDS PharmaServices, Inc. proprietary algorithm in an effort to select the proper cell plating density for each cell line. The performance metrics include coefficients of variation (CVs) of Controls for the cell plating data and Data Set Mean CVs for assessing replicate propinquity (i.e., how close the replicates are to each other). The performance of the automation system and our algorithm for this project produced data of superior quality.
Edge effects—the tendency of the contents of wells in the outer rows and columns of a multi-well microplate to vary from wells in the interior of the plate—are due to interactions of the contents of the wells of the microplate with the laboratory or storage environment. Evaporation is rapid at the corners and edges while wells near the center are protected from change. Hygroscopic liquids (e.g., dimethyl sulfoxide (DMSO)) can rapidly absorb water from the environment and the tendency towards hydration is greatest at the edges and corners. The gradient between the contents of a plate and the environment is greatest at the edges of the plate and the fastest changes occur at that steep gradient.
Innovative MicroClime® lids are compatible with standard automation and unlimited lid/de-lid When filled with DMSO filled they can reduce DMSO hydration rate; lower DMSO evaporation rate; reverse hydration and preserve small (nL) dispensed drop volumes When filled with water and on assay plates they reduce “edge effects” and enable low-volume aqueous assays to be extended over multiple days