title: Optimization of Compound Plate Preparation to Address Precipitation Issue in Mammalian A549 Cytotoxicity Assay.
abstract: This study illustrates the optimization of low-volume dispensing on a liquid handling system (LHS) to overcome the precipitation of compounds in the mammalian cytotoxicity assay with low dimethyl sulfoxide (DMSO) tolerance. All compounds at AstraZeneca Bangalore are tested in the mammalian cytotoxicity assay. In order to maintain the DMSO levels, serially diluted plates were prepared in DMSO/water. It was observed that some of the compounds precipitated. The IC50 data for such compounds were therefore erratic. To circumvent the problem of compound precipitation, the LHS was optimized to dispense low volumes (<1 µL). The plates were serially diluted using neat DMSO. Since the dilution was done using neat DMSO, there were no issues with precipitation. The serially diluted sample (0.5 µL) from the plate was stamped onto the assay plate to give the desired DMSO concentration. No significant differences in IC50 data were observed for 1 µL dispenses made from DMSO/water and 0.5 µL dispenses from neat DMSO for the samples with no precipitation issues. These data therefore gave us the confidence to switch over to 0.5 µL dispenses for the cytotoxicity assay to address the precipitation issue. However, precipitation of samples in the assay buffer is beyond the scope of this discussion.
authors.affiliation: Compound Management, Department of Biosciences (Microbiology), AstraZeneca India Pvt. Ltd., Hebbal, Bangalore, India vijayashree.achar@astrazeneca.com vijayashree75@gmail.com.
authors.forename: Vijayashree Gauribidanur
authors.initials: VG
authors.lastname: Raghavendra Achar
authors.affiliation: Compound Management, Department of Biosciences (Microbiology), AstraZeneca India Pvt. Ltd., Hebbal, Bangalore, India.
authors.forename: Shubhada Pramod
authors.initials: SP
authors.lastname: Barde
authors.affiliation: Compound Management, Department of Biosciences (Microbiology), AstraZeneca India Pvt. Ltd., Hebbal, Bangalore, India.
authors.forename: Meenakshy Venkatesh
authors.initials: MV
authors.lastname: Mallya
authors.affiliation: Compound Management, Department of Biosciences (Microbiology), AstraZeneca India Pvt. Ltd., Hebbal, Bangalore, India.
authors.forename: Disha
authors.initials: D
authors.lastname: Awasthy
authors.affiliation: Compound Management, Department of Biosciences (Microbiology), AstraZeneca India Pvt. Ltd., Hebbal, Bangalore, India.
authors.forename: Chandan
authors.initials: C
authors.lastname: Narayan
mesh_terms: D002460:Cell Line; D011232:Chemical Precipitation; D003584:Cytological Techniques / Q000379:methods*; D004847:Epithelial Cells / Q000187:drug effects*; D015386:Hazardous Substances / Q000737:chemistry*; D006801:Humans; D020128:Inhibitory Concentration 50; D012995:Solubility; D014116:Toxicology / Q000379:methods*
chemical_list: D015386:Hazardous Substances
keywords: Biomek FX liquid handler; compound management; low-volume dispense; mammalian A549 cytotoxicity assay