Researchers see the benefits of streamlining accurate serial dilution of stock solutions of compounds.
As the cost of drug development escalates there is a growing requirement to accelerate the R&D cycle and eliminate unwanted off-target drug effects earlier. One way of achieving these goals is through assay miniaturization and production of assay ready plates, which leads to higher throughput and reduces reagent and compound cost. Many assay protocols require the accurate serial dilution of stock solutions of compounds. Streamlining this process using automated liquid handlers is beneficial.
Figure 1: Top plate: source plate of 96 compounds; Middle plate: schematic demonstrating the source plate layout after each compound has been diluted into four stock solutions; Bottom plate: schematic of one assay-ready plate containing 32 compounds. (Source: TTP LabTech)
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In screening laboratories, compounds are routinely diluted from a single stock solution in a stepwise, serial manner across microplates using constant volumes. More recently a two-stage method of generating serial dilutions has become popular. The initial stage involves the preparation of multiple stock solutions differing 30-100-fold in concentration. This is then followed by the transfer of different nanoliter volumes of the stock solutions into a second assay microplate to create the ‘assay-ready’ concentration-effect curve.
The mosquito low volume liquid handler from
TTP LabTech combines a disposable tip system with a positive displacement pipette to ensure zero cross contamination. This instrument can accurately aspirate and dispense a wide range of liquid types and is capable of pipetting volumes from 1.2 µL down to 50 nL with no washing required. The liquid handling capability of mosquito has been expanded by combining the instrument with the newly developed bulk dispenser module.
The mosquito bulk dispense head module allows high volume (10 µL), non-contact dispensing from eight independent channels into plates located on the mosquito deck. This enables high and low volume pipetting steps to be carried out within the same protocol through a common user interface. Mosquito, in conjunction with the newly developed bulk dispense module, is capable of performing two-stage compound dilution and this article describes the two-step dilution process of 96 compounds in 384-well microplates using an integrated pipetting protocol.
Materials and Methods
A two-stage approach was used to generate the concentration-effect curves. In the initial stage, compound stock solutions in a 384-well source plate were serially diluted to provide four stocks of 10 mM, 0.316 mM, 10 µM, and 316 nM for each compound. In the subsequent step, aliquots of each of the four stocks were transferred from the source plate into three 384-well plates to create assay-ready, concentration-effect plates that each contained 12-point half-log dilution series for each compound, with each plate containing 32 compounds.
Figure 2: Fluorescence intensity was used as a measure of the volumetric accuracy of the mosquito direct dilution method versus a manual serial dilution method. (Source: TTP LabTech)
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Preparation of Step Dilutions
In order to prepare the step dilutions, stock transfer was performed using low volume micropipettes and diluent was added using the bulk dispenser module. All stock solutions of compounds were dissolved in 100% DMSO. In the first instance 35 µL of each compound at the top stock concentration was placed in columns 1, 5, 9, 13, 17, and 21. Mosquito was then used to transfer 1,108 nL of all compounds into the adjacent column (2, 6, 10, 14, 18, and 22, respectively). The bulk dispense module was used to add 34 µL of 100% DMSO to these columns, causing instantaneous turbulent mixing in the process and diluting the compound 31.6-fold, that is by 1.5 log units. By repeating this process twice, four different stock solutions of 10 mM, 0.316 mM, 10 µM, and 316 nM, all diluted in 100% DMSO, were produced and these were used as the source plate for Stage 2 (Figure 1, middle plate). The overall time for this process was approximately four minutes.
Direct Pipetting for Creation of Concentration-Effect Curves
During the second half of this procedure, 500 nL, 158 nL, and 50 nL of the highest compound stock was transferred from the source plate into columns 1 to 3 of a new 384-well assay plate. The volume in each well was made up to 500 nL using 100% DMSO. This was repeated for the three other stock solutions creating up to a 12 point half-log concentration-effect curve for each compound. Alternatively the last dilution can be omitted in favor of a DMSO solvent blank, which would result in an 11-point concentration-effect curve that spans almost six log points. This process was then repeated for the next 16 compounds in the other half of the assay plate (Figure 1, bottom plate, page 26). The preparation of each plate was about 3.5 minutes and it was repeated three times in order to complete the dilution of all 96 compounds into three 384-well assay-ready plates.
Figure 3: A concentration-effect curve of vinblastine on cell number: serial dilutions of vinblastine were created using either the mosquito direct pipetting protocol or via a manual method. (Source: TTP LabTech)
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Results
In order to compare the accuracy of the mosquito integrated pipetting method, manual dilution experiments were performed in parallel. Stock solutions of 1 mM fluorescein were used in the dilution protocol, and the accuracy determined by measuring the fluorescent intensity per well. The results demonstrated that the volumetric accuracy was very high and was comparable to the manual method (Figure 2). The direct pipetting method eliminated the compounding of serial dilution errors. Furthermore, the direct dilution method resulted in improved repeatability and reduced errors over traditional automated serial dilutions where the mixing can be inefficient. In all columns, the CVs were less than 8%.
The two-stage direct pipetting dilution method was also evaluated in microplates that contained live cell cultures. The use of adherent cell cultures is prevalent in drug screening and often requires addition of test compounds after plating, which precludes the use of assay ready plates. Direct pipetting should overcome this problem and, moreover, does not require the removal of the growth medium, a procedure that can be very disruptive to some assays. HeLa cells were seeded in a 384-well plate and left overnight to adhere before being treated with varying concentrations of the G2/M blocker, vinblastine, for 22 hours. In columns 1 to 12 of the microplate, the automated direct dilution method was used to prepare the vinblastine concentrations, whereas a manual method was used to serially dilute vinblastine in columns 13-24. Prior to counting, cells were ethanol fixed and then labelled with 10 µM propidium iodide before the cell number was quantified on the Acumen eX3 laser scanning microplate cytometer (TTP LabTech) using the 488 nm laser. As Figure 3 demonstrates, the automated direct dispensing of reagents into assay plates containing live cells was successfully used to generate biological concentration-effect responses for vinblastine treatment on cell number. The curves obtained were comparable to those that were obtained with the manual serial dilution method demonstrating that automated serial dilution can be undertaken in plates containing live cell cultures.
Conclusion
Two-stage dilution on a single platform eliminates integration and provides rapid production of assay-ready plates. Mosquito, in conjunction with the newly developed bulk dispenser module, is capable of performing two-stage serial dilution and can set up as many as 370, 12- point, six-log, concentration-effect curves each hour. Using this methodology the compounding errors that are often seen with serial dilution protocols are eliminated and the stock dilution in DMSO and direct pipetting into assay plates improves compound stability down the series. Finally, through the direct dispensing of reagents/compounds into assay plates containing cells, concentration-effect curves can be generated without serial dilution in an intermediate plate.
Joby Jenkins is the product manager for mosquito at TTP LabTech. He has 10 years of industrial experience in industrial research and development, focusing on project management and sales and marketing.