title: Adherent cell assay results affected by variable z-position mixing.
abstract: We demonstrate that modifying mixing dynamics after addition of organic solute into aqueous buffers dramatically affects cell morphology and protein expression. Variable z-position (VZP) or varying the height of aspiration and dispense positions during mixing eliminates artifactual effects. Here, we tested 4 adherent cell types and show effects of VZP on quantitative imaging, protein expression, viability, and morphology. The result: The quantitation of cytoplasmic fluorescence within the fields of interest of the phalloidin-actin stain assay improved by 47% and fluorescence variability emitted by cells expressing green fluorescence protein (GFP) fusion proteins decreased by 15%. Assays that perform measurement by averaged reading of the entire well are somewhat susceptible. For example, protein production decreased 8% on the hypoxia response element (HRE)-luciferase assay. VZP did not affect quantitative cell viability, deviate the half maximal effective dose concentration (EC(50)) values or alter expected curve patterns. VZP is a valuable systematic process for cellular assay workflows as it efficiently folds organic solute into the aqueous solution.
authors.affiliation: Research and Automation Technologies, Amgen, Thousand Oaks, California 91320, USA. nelsonc@amgen.com
authors.forename: Nelson
authors.initials: N
authors.lastname: Carramanzana
authors.forename: Sandra
authors.initials: S
authors.lastname: Ross
authors.forename: Gloria
authors.initials: G
authors.lastname: Biddlecombe
authors.forename: Chi-Hwei
authors.initials: CH
authors.lastname: Lin
authors.forename: Michael
authors.initials: M
authors.lastname: Johnson
mesh_terms: D001331:Automation; D001562:Benzimidazoles; D001681:Biological Assay / Q000379:methods*; D002448:Cell Adhesion; D002459:Cell Fusion; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects; D004121:Dimethyl Sulfoxide; D016650:Fluorescein-5-isothiocyanate; D005456:Fluorescent Dyes; D049452:Green Fluorescent Proteins / Q000737:chemistry / Q000235:genetics; D006367:HeLa Cells; D006801:Humans; D009363:Neoplasm Proteins / Q000096:biosynthesis / Q000235:genetics; D010590:Phalloidine; D010784:Photomicrography; D012997:Solvents
chemical_list: D001562:Benzimidazoles; D005456:Fluorescent Dyes; D009363:Neoplasm Proteins; D012997:Solvents; D049452:Green Fluorescent Proteins; D010590:Phalloidine; D016650:Fluorescein-5-isothiocyanate; C017807:bisbenzimide ethoxide trihydrochloride; D004121:Dimethyl Sulfoxide