(-)-Blebbistatin ((S)-(-)-Blebbistatin) is an S enantiomer of blebbistatin. It is a potent and selective myosin II inhibitor with IC50 ranging from 0.5 to 5 μM.

METHODS: Mouse hepatic stellate cell HSCs were treated with (-)-Blebbistatin (12.5-50 µM) for 2 h and cell morphology was observed using microscopy.
RESULTS: When treated with (-)-Blebbistatin for 2 h, the cells became smaller, acquired more dendritic morphology, and had fewer stress fibers and lamellipods. [1]
METHODS: Porcine TM cells were treated with (-)-Blebbistatin (25-100 µM) for 2 h. The expression levels of target proteins were detected using Western Blot.
RESULTS: There was no difference in MLC phosphorylation status between control and drug-treated samples. [2]

Blebbistatin completely relaxes both KCl- and carbachol-induced rat detrusor and endothelin-1-induced human bladder contraction in a dose-dependent manner. Pre-incubation with 10 μM blebbistatin attenuates carbachol responsiveness by 65% while blocking electrical field stimulation-induced bladder contraction reaching 50% inhibition at 32 Hz[4].

Inhibition of Cox Activity: Inhibition of Cox-1 activity is measured by monitoring oxygen consumption during the conversion of arachidonic acid to PGs using a Clark-type O2-electrode. The reaction mixture contains ～0.2 units Cox-1 in 100 μL of microsome fraction derived from ram seminal vesicles as a crude source of Cox-1 (specific activity 0.2–1 units/mg protein) or 0.23 units of recombinant human Cox-2 (specific activity 43 units/mg protein). For calculation, the rate of O2 consumption is compared with a DMSO control (100% activity). Piroxicam, a nonsteroidal anti-inflammatory drug, is used as positive inhibitory substance for Cox-1 activity with an IC50 of 0.35 ± 0.05 μM (n = 2). Alternatively, nimesulide, a Cox-2 specific inhibitor, inhibits Cox-2 activity by 52 ± 5.7% (n = 2) at a concentration of 50 μM.

Freshly isolated HSCs are replated on 96-well plate. At day 3, medium is replaced by serum-free medium and cells are starved overnight, treated with or without blebbistatin (25 μM) for 2 h followed by stimulation with platelet-derived growth factor-BB (20 ng/mL). After an overnight incubation, the WST-1 cell proliferation assay are performed[3].