A critical requirement for multiplexing agonist and antagonist screens with the calcium assay is whether the reagent dispensing system is adequate in the fluorescence kinetic plate reader to function in HTS mode. Previous versions of fluorescence kinetic plate readers either had only one pipette head or lacked sufficient tip wash modules to rapidly remove residual compound from the pipette tips. The detection of agonist responses occurring in few seconds to a few minutes requires addition of test compound solution inside the instrument immediately after the baseline recording. In addition, the final DMSO (the compound vehicle) concentration in the assay plate usually must be <0.5% (v/v) to avoid the adverse solvent effect. Thus, the amount of compound solution added to the cells in the assay plates should be ≤200 nL for 384-well plates containing 40
μL of total volume or ≤25 nL for 1,536-well plates containing 5
μL of total volume. To handle such small volumes of compound solution rapidly and in parallel, the 384- or 1,536-well pin tool is clearly more appropriate than pipette tips because it can quickly and accurately transfer a fixed amount of compound solution (from 10 to 500 nL dependent on the size of pins selected).22
After pin tool addition of compounds and while the agonist response of compound is recording, a second pipette head with exchangeable tips is mounted to the moving head unit that then transfers the EC90
amount of agonist solution to the assay plate for the recording of antagonist response of the compounds. Use of 2 pipette heads for separate dispensing of compounds and agonist avoid the cross contamination of these 2 reagents in their source plates/reservoirs.
In order to quickly mix the compound DMSO solution with the buffer in wells of the assay plate, the pin tool “trituation” (rapid vertical movements within 1–5
mm distance adjustable by users) is used in combination with the quick rotational movement of the assay plate. For mixing the agonist solution with the buffer in assay plate, a ratio of 1:4 to 1:2 agonist solution (eg, 1–3
μL/well of agonist in a 1,536-well plate) has been used in combination with the rapid assay plate movement to facilitate mixing. As these agonist and antagonist responses of GPCR ligands are usually very rapid, both compound DMSO solution and EC90
agonist stimulation solution must be added to all wells in an assay plate simultaneously while the plate being read kinetically in the instrument. Our results have demonstrated that the FDSS7000 instrument has sufficient fluidic systems and mixing functions to accommodate the needs of multiple dispensing protocols, rapidly switching pipette heads and rapid tip washing to limit compound carryover.
The pipette tips on the 1,536-well head and the pins on the pin tool head need to be washed and cleaned within 1 to 2
min after each use for the continuous measurement. The choices of right solvents for the tip/pin wash are important for eliminating the compound carryover between plates. We found that one solution wash (eg, either DMSO or water) was insufficient to remove the residual compounds from tips and pins (data not shown). It can be explained by the diversity of compounds in the collection—some of them are lipophilic and others are hydrophilic. A combination of one DMSO bath and one water bath for wash with a brief sonication is an effective method to remove residual compounds on the tips or pins. A tip-blotting step in which the tips touch a layer of filter paper in a module after the tip wash steps wipes away the remaining liquid on the tips or pins. This cycle of tips washes, tip blotting, and pipetting head change can be completed within 2
min in this instrument and can be adjustably controlled by the operation software. Thus, this improved liquid-handling system in this kinetic plate reader enables the rapid and complex protocols that are needed for sequential agonist and antagonist screens in a single assay plate. However, we have observed the high irreproducible hit rate from the antagonist screen, indicating the uneven dispensing of agonist by the 1,536-well pipettor though the CV of dispensing may be still in the acceptable range of 5%–10%. Thus, the performance of this 1,536-well pipettor should be further optimized and improved by the manufacturer.
In the miniaturized assay format, it is a challenge to rapidly and completely mix the dispensed compound solution or agonist solution with the existing solution in a assay plate during a kinetic measurement because of the narrow and tall well dimension, ~2 (w)
8 (H) mm for a standard 1,536-well plate. Gravity and diffusion are the main forces for the quick solution mixing in the 96-well plate assay but it takes longer time for the solution to be mixed in the higher well density plates.23
The higher gravity of compound DMSO solution may cause the compound sinking quickly to the bottom of plates resulting in a transit higher local concentration near cells attached on the bottom of assay plates.16
This high local concentration of compound resulting from insufficient mixing may lead to inaccurate compound activity. Or a concentration gradient of the added solution may be formed due to insufficient mixing in the higher well density assay plates that affects the accuracy of compound activity. In this new instrument, the combination of rapidly vertical pin movement and assay plate shaking functions can create effective and rapid mixing of the compound DMSO solution with the assay plate buffer while the plate is continuously read and recorded. We found that the local high concentration of compounds due to insufficient mixing can be avoided by using these functions in this instrument.
The main difference between GPCR agonist mode and antagonist mode in compound screens is the addition of an EC90
amount of a known agonist prior to recording the antagonist response. Thus, the antagonist response can be sequentially measured in the same assay plate after the agonist response is read and recorded by a rapidly addition of EC90
amount of a known agonist. A multiplex screen assay for GPCR agonist, potentiator, and antagonist in 384-well plate format was reported using a previous version FDSS6000 instrument.24
A dye removing step was required in that assay for the reduction of fluorescent background that could significantly decrease the screen throughput. Recent advances in the liquid-handling system in this kinetic plate reader in combination with the no-wash calcium dye contributed to the development of this multiplex agonist and antagonist assay in the 1,536-well plate format that simplifies the assay procedure, increases the screen throughput, and thus should be suitable for HTS. The fluorescence quenching dye in the no-wash calcium dye kit helps to eliminate the cell wash step after dye loading that enables an addition-only assay format for the calcium assay without affecting the pharmacology of agonist and antagonist. Our results from the validation tests using the muscarinic acetylcholine M1
receptor transduced cell line also demonstrated that the potencies of 5 known agonists and antagonists were similar in both the single mode and the sequential assay mode in the 1,536-well plate format.
In conclusion, the FDSS7000 fluorescence kinetic plate reader, a new generation model, has been equipped with a new liquid-handling system and sensitive optical detection train and can be used for more complicated and automated assay protocols. The multiple pipetting heads and tip washing modules are major enhancements that greatly improve cleaning efficiency and transfer times for pipette tips and pins as well as reducing the possibility of compound carryover. Our results also demonstrated that this fluorescence plate reader can be used for sequential screens of compounds for agonist and antagonist activities in a single 1,536-well assay plate. We believe that these characteristics will allow the full automation of such protocols in qHTS format, a future goal of the NCGC that should further improve the data quality in the primary compound screen.