In the field of pharmaceutical research, much depends upon the measurement of specific drug activity as compared to other available molecules. In order to achieve standardized reports, the level of activity of the drug molecule against a target is expressed in terms of the IC50 and EC50 values, which are the half maximal inhibitory and half maximal effective concentrations, respectively, of the drug.
These are obtained by dose-response curves, which are in turn based on the concentrations of the drug dissolved in a solvent. The solvent used is most commonly DMSO as this has become the standard.
Currently, the drug management teams are given the solutions of the compound to be tested dissolved in DMSO, and the targets need to be selected from many different microplates.
This process of selection is called hit-picking. The targets themselves are chosen after one single high throughput screening (HTS) procedure, which yield concentration data.
In this context, the Fluent system from Tecan may be said to have changed everything in automated laboratory processes. Fluent is designed to meet each laboratory’s special needs, unique to different applications. It not only offers greater capacity but performs analyses at higher speed. The increased throughput and independent operation makes for a more productive process and increased confidence in the results.
Figure 1: The Fluent compound management solution. A Fluent 1080 equipped with an eight-channel Flexible Channel Arm, a Multiple Channel Arm and a Robotic Gripper Arm is shown. A Carousel is integrated onto the right hand side of the instrument.
Materials and methods
Fluent Laboratory Automation
The Fluent system from Tecan is the latest automated compound management system for liquid handling. As shown in Figure 1, Fluent allows pipetting of solutions in a rapid and accurate manner using either the Flexible Channel Arm (FCA) which has eight channels or the Multiple Channel Arm (MCA).
Its Dynamic Deck™ application provides increased capacity as well as allowing more to be done within the same time. This deck lets the user integrate several modules from the Tecan range, such as a Carousel to store all kinds of consumables (including carriers for troughs, single-use tips and microplates, all of which may be stacked on the worktable) as shown in Figure 2.
The adapters on the MCA may be interchanged automatically, from 96 to 384 channels, without interrupting the process run or changing the protocol. The use of independent arms specific to each task mean that processes can be coordinated to run in parallel, speeding up runs and shortening schedules, while increasing the efficiency.
Figure 2: Typical deck layout for the Fluent compound management solution. Microplates can be stored up to six deep on one deck segment.
Each component of the Fluent compound management system is dealt with in more detail below:
- The FCA has eight channels with either reusable or single-use steel tips fixed to the ends. This enables each test tube or well to be used singly, ensuring ease of distribution of samples and controls, as well as of serial dilution procedures.
- The MCA allows instantaneous automated shifting of adapter plates from 96 to 384 channels during a single run, so that pipetting can be extended to a higher number of wells or tubes, whether for reagent distribution or plate replication.
- The adapter plates are varied:
- The COMBO adapter has 384 single-use tips in a box, which may be chosen in vertical arrays of 16 or horizontal arrays of 24, to allow parallel pipetting to be performed.
- The Non-COMBO adapter has 384 disposable tips to allow for parallel pipetting.
- The Extended Volume Adapter (EVA) can pipette up to 500 μl by mapping four channels into one, using MCA 96 single-use tips. It can pick up multiple columns or rows of either 8 or 12 disposable tips, respectively, from the tip box. Other tip boxes of differing shapes may also be used, such as one with 4 columns by 6 rows, or 12 columns by 8 rows.
- The Non-EVA adapter is used when four-to-one channel mapping is unnecessary, to pipette samples up to 125 μl in parallel with MCA 96 single-use tips. It may use columns of 8 or rows of 12 such tips from the special box but can also use tips from other tip boxes as above.
- The adapter for using 96 out of 384 tips, which can pipette up to 125 μl using 96 tips as a quadrant from the MCA 384 tip box. It can pick up as many columns of 8 or rows of 12 as required, at the same moment.
- The Robotic Gripper Arm (RGA) is essential to move both plates and the consumables between the various stages of processing, such as the storage devices, the worktable and other integrated parts of the process run, without having to pause in between.
The Fluent system can work with either microplates or tubes, including those which are 2D barcoded, and various combinations of labware may be used depending on the format selected. Thus, the IC50 plate may have samples pipetted into rows of tubes previously specified, or of wells in rows of 96, 384 and 1536 as per requirement.
Screening plates may be another option for sample distribution, using just one or an entire plate of samples for hit-picking. When the experiment utilizes a high throughput single concentration set of samples for hit-picking, less than 2% (or four samples per plate) are normally picked, which means that the user should be able to access a number of plates at high speed.
The Fluent system is thus unique in allowing IC50 plates to be set up with either the FCA or the MCA arm. The FCA is filled with liquid and so allows DMSO to be utilized as the system fluid for washing up fixed tips, backfilling or preparing diluent plates faster.
This part of the article discusses two methods of hit-picking using serial and double serial dilution, respectively.
Method 1: Use of the FCA to hit-pick from the worklist, and subsequent serial dilution using the MCA
In this method, a 384-well plate is used in 24 steps, with 1.5 dilution at each step, to achieve dilution of 14 compounds and 2 controls.
The first step is to prepare a worklist with each microplate identified by the barcode, as well as the hit-picking sites within each plate by its coordinates such as A3 and D9. This is available to the FluentControl™ software while the protocol is being set up.
The Carousel is loaded with the source plates and the dilution plates so that the Robotic Gripper Arm can shift plates identified by the Carousel’s barcode reader to the worktable, once the worklist starts, first having removed the plate lids if present.
The worktable trough is filled with 100% DMSO and most of the compounds to be tested are dissolved in this solvent as well, which is also the solvent used for serial dilution. The MCA is used to lift the COMBO plate as well as 384 disposable tips, of 125 μl capacity. It then sucks up 80 μl of solvent and releases it into an empty plate with 384 wells, discharging the tips only.
Figure 3: Preparation of a 384-well dilution plate for serial dilution.
The steel tips fitted to the FCA suck up the volume required, such as 20 μl, from the previously coded locations in the worklist, to the defined plates. An example is plate barcode xxx, position G5, to plate barcode xy, positions B7 and D2.
This may be seen in Figure 4. Seven of these channels can hit-pick specified samples and the eighth one aspirates the same volume of a control from the worktable. The specified volumes of each sample are then released into column 1 of a dilution plate with 384 wells, in this experiment the control being dispensed into A1 and the targets into B1 to H1.
These have been previously filled with 80 μl of DMSO and the compounds are mixed in completely. This is done over again for the next seven samples using a control, with the use of wells 11 and J1 to P1, in column 1 for the control and target compounds, respectively. Thus, the plate is finally converted into a 384-well dilution plate with 14 targets and two controls.
Figure 4: The Flexible Channel Arm aspirating hits from two predefined microplate positions in parallel.
The COMBO plate can also be used to take up the first column of single-use tips from the first column, 16 in number, of a 50 μl MCA 384 single-tip use box, by placing the plate on the nested 384 plates rather than on the active carrier module, which ensures that this pick up is performed on shifted tips.
20 μl of each hit and of the control is sucked up from column one of the dilution plate and released into the wells of column 2 of the same plate, filled with DMSO, which makes it a 1:5 dilution. This is then thoroughly mixed. Repetition of this dilution process continues, from the second to the third column and so on, until all 24 columns of the plate are filled.
Figure 5: The Multiple Channel Arm picks up a single column of 16 disposable tips from a box of 384 tips.
Figure 6: Plate layout and pipetting scheme for the serial dilution method: 20 μl of 14 compounds and two controls are diluted across a complete 384-well plate in a 24-step series of 1:5 dilutions.
At this point, the MCA sucks up 20 μl from column 24 wells. This is to make sure all wells contain 80 μl of diluted compound. The solution is discarded, and the tips are placed into an empty box which contained MCA 384 disposable 50 μl tips, on the worktable. This frees the workflow to proceed and repeat the entire process to achieve the next serial dilution.
Method 2: Use of the FCA to hit-pick from the worklist, and subsequent double serial dilution using the MCA
While closely resembling method 1, the final dilution is 1:10 and 28 compounds with 4 controls are used in a 384-well microplate in parallel.
The MCA first attaches the COMBO plate and 125 μl MCA 384 single-use tips. It sucks up 90 μl of DMSO from the worktable trough and releases it into an empty dilution plate with 384 wells. The tips are released but not the plate.
The FCA has steel tips to suck up the volume required, such as 10 μl, from the coded locations on the worklist such as plate barcode xx, position G5 to plate barcode xy, position B7 and D2. Just as in method 1, seven of the FCA channels pipette up specified targets while the eighth removes a corresponding volume of a control from the worktable.
The required amounts of hits and control compound are released into column 1 of the 384-well plate, the control in well A1 and the target compounds in wells B1 to H1 and mixed thoroughly with the 90 μl of solvent with which each well is filled. This is done again three times. The second batch of targets and controls are again pipetted into the same column, J1 to P1 and I1, respectively.
The third and fourth batches are dispensed into wells A13 to P13 of column 13. The end result is a 384-well plate with 28 hit-picked samples and four controls. The tips for the next few steps are obtained in the right form as follows: the first 16-tip column of a box of 50 μl MCA 384 single-use tips is picked up by the MCA using the COMBO plate.
This is put down into another empty MCA 384 single-use tip box in column 13. These boxes are called the ‘first’ and ‘second’ boxes for clarity. The MCA now picks up the second 16-tip column from the first box and puts them into column 1 of the second box. These two columns from the second box are now picked up by the MCA in this array.
The MCA is now used to suck up 10 μl volumes of each target compound from column 1 and 13 of the plate, in parallel, and to put them into columns 2 and 14 whose wells already contain diluent and mixed to obtain a 1:10 dilution. This dilute solution is now sucked up and dispensed into the next 2 columns (3 and 15 respectively), and the process goes on repeatedly until all the columns are filled. This is depicted in Figures 12 and 13.
At the end, the MCA is used to tidy up the worktable to the original arrangement for further use in subsequent repetition of this serial dilution procedures. It picks up the tips from column 1 and 13 of the second box and puts them into column 1 and 2 of another box which is empty.
This article describes how compounds and controls are diluted in serial and double serial dilution using all columns of a 384-well microplate. In the first process, 14 compounds and 2 controls are diluted in 24 steps. In the second process, 28 compounds and 4 controls are diluted in 12 steps.
These processes and steps may be modified in accordance to the specific requirements of each laboratory. Thus, 42 compounds and 6 controls could be subjected to triple serial dilution in 8 steps, or 56 compounds and 8 controls to quadruple serial dilution in 6 steps.
The type of serial dilution determines the number of hits to be picked and the target columns. Thus, for a 384-well plate, the following are the target columns:
- Single serial dilution – column 1
- Double serial dilution – columns 1 and 13
- Triple serial dilution – columns 1, 9 and 17
- Quadruple serial dilution – columns 1, 6, 13 and 19
The array of tips must also be adapted to the dilution using the same technique of picking up one column from a full MCA 384-tip box and discharging it into another empty box in the right column. This is repeated until the required array is ready. The principle is that the target columns in the empty tip box and in the hit-picking box are the same, as in the above list.
In the last step, the controls can be easily changed to the number that fits the laboratory protocol, using the solution in the worktable trough rather than an unknown compound and adding it as part of the whole process along with hit-picking.
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