RESUMO
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.
Assuntos
Bioensaio/métodos , Adesão Celular , Automação , Benzimidazóis , Fusão Celular , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Dimetil Sulfóxido , Fluoresceína-5-Isotiocianato , Corantes Fluorescentes , Proteínas de Fluorescência Verde/química , Proteínas de Fluorescência Verde/genética , Células HeLa , Humanos , Proteínas de Neoplasias/biossíntese , Proteínas de Neoplasias/genética , Faloidina , Fotomicrografia , SolventesRESUMO
A series of (4-piperidinylphenyl)aminoethyl amides based on dipeptide anilines were synthesized and tested against cathepsin K, cathepsin L and cathepsin B. These new non-covalent inhibitors exhibited single-digit nM inhibition of the cysteine proteases. Compounds 3 and 7 demonstrated potency in both mouse and human osteoclast resorption assays.
