High sensitivity β galactosidase assay kit

M17 cells were placed in 48 wells and cotransfected with 0.05 μg of the firefly luciferase report vector, 0.4 μg of hU6-hsa-miR-124-CMV-GFP or hU6-hsa-miR-Scramble-CMV-GFP, and 0.01 μg of pMIR-REPORT β-gal control plasmid (Ambion, Austin, TX, United States) for normalization of transfection using the Lipofectamine 2000 reagent according to the manufacturer’s protocol. After 24 h, the luciferase activity was measured using the Bright-Glo luciferase assay system (Promega), and the β-galactosidase activity were measured using the high-sensitivity β-galactosidase assay kit (Agilent Technologies, Palo Alto, CA, United States). The luciferase activity was normalized against the β-Gal activity for the same cells. The experiments were performed in triplicate.

The SOX2 promoter connected downstream to the Luciferase gene was purchased from SwitchGear Genomics, a pCMV-LacZ vector was purchased from Clontech, and an expression vector of pcDNA3.1 harboring HOXA5 cDNA was established in our laboratory. Luciferase assay was performed using Luciferase Assay System (Promega) according to the manufacturer's protocol, and β-galactosidase assay as an internal control was performed using a High Sensitivity β-Galactosidase Assay Kit (Agilent Technologies) according to the manufacturer's protocol.

LacZ activity was evaluated using chlorophenol red-β-D-galactopyranoside as described for the High Sensitivity β-galactosidase Assay Kit (Agilent Technologies). Cell lysates were incubated with the substrate usually for 30 min at room temperature. Absorbance was measured at 595 nm.