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A Microscopic Phenotypic Assay for the Quantification of Intracellular Mycobacteria Adapted for High-throughput/High-content Screening
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Confocal-based method for quantification of diffusion kinetics in microwell plates and its application for
Ok Ryul Song1, Tae-Hee Kim, Xavier Perrodon
1Screening Technologies and Pharmacology Department, Institut Pasteur Korea, Gyeonggi-do, South Korea.
Journal of Biomolecular Screening
|January 21, 2010
Summary
Efficient mixing in microplates is crucial for high-content/throughput screening (HC/TS). Rapid plate shaking and bulk buffer addition are effective methods for ensuring fast and gentle mixing of compounds and cell culture medium.
Area of Science:
- Biochemistry
- Cell Biology
- Assay Development
Background:
- Efficient mixing is critical for high-content/throughput screening (HC/TS) assays.
- Noncontact nanoliter liquid handlers present unique mixing challenges.
- Biological kinetics necessitate rapid mixing within minutes, while preserving cell layers.
Purpose of the Study:
- To introduce a method for quantifying nanoliter drop diffusion in microplates.
- To evaluate the impact of various mixing methods on diffusion in 384-well plates.
- To identify optimal mixing strategies for HC/TS.
Main Methods:
- Developed a novel method to quantify drop diffusion independent of buffer or liquid handler.
- Tested mixing efficiency using nanoliter drops of DMSO in aqueous buffer.
- Assessed effects of rapid plate shaking, buffer addition, and bulk addition on mixing.
Main Results:
- Rapid plate shaking and additional buffer addition proved most efficient for HC/TS.
- Plate shaking efficiency is constrained by assay volume.
- Bulk addition demonstrated fast, efficient mixing without adverse cellular effects.
Conclusions:
- Bulk addition offers a simple, fast, and inexpensive mixing solution adaptable to various platforms.
- This method facilitates gentle yet effective mixing for HC/TS.
- Optimized mixing protocols enhance screening assay reliability and speed.
