| Assay Method Information | |
| | Assaying Nonmuscle Myosin II |
| Description: | Cytokinesis is a cell-based assay to assess nonmuscle myosin II function, performed using COS7 cells. The assay is run in a 96 well plate, with 2,000 cells plated per well. Total incubation time is 48 hours, with 24 hours of treatment with the compound of interest. Prior to treatment with small molecules, a two-fold dilution series of compound solutions is prepared in DMSO. Starting concentrations are determined based on compound solubility. Compound solutions are further diluted in DMEM medium to a final DMSO concentration of 2% (50-fold dilution). Subsequently, 100 μl diluted solution is transferred to each well of the plate containing 100 μl of cell culture (achieving 1% final DMSO concentration). All measurements are carried out in triplicate. Cells are stained by fluorescein diacetate (6 μM), a cell viability dye, the cell-permeant Hoescht33342 (10 μM) and the membrane-impermeant propidium iodide (4 μM) to label all nuclei and those belonging to dead cells, respectively. Dye solutions are replaced by fresh media after 10 minutes of incubation. An INCell Analyzer 6000 is used for imaging. The signal of interest is the ratio of nuclei to cell numbers, as nonmuscle myosin II inhibition prevents cellular blebbing, resulting in multinucleated cells. Cytotoxicity is also assayed and quantified as the ratio of dead nuclei to total nuclei. 20 μM para-aminoblebbistatin in 1% DMSO serves as the positive control and 1% DMSO alone is the negative control. Half maximal effective concentration (EC50) is determined by fitting the 6-point dose-response data to the Hill equation. Small molecules showing no observable inhibition are reported as inactive. |
| Affinity data for this assay | |
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