Glomax multi jr single tube multimode reader

Luciferase activity was measured in U87 transduced cells using dualluciferase reporter assay (Promega, Madison, WI). U87^Luc and U87^mock were seeded in 6-well plates and allowed to reach 70% confluency. After removing cell medium and washing once with PBS, the cells were lysed in 500 μL 1X passive lysis buffer (PLB) for 15 min at room temperature with gentle shaking. Subsequently, 10 μL of PLB lysate was mixed with 50 μL LARII (luciferase substrate) and firefly luciferase activity was determined using GloMax-Multi Jr Single-Tube Multimode Reader (Promega, Madison, WI). Mean luciferase luminescence was 2.1×10⁷ relative light units (RLU) per 10⁶ U87^Luc cells (± SD 6.4×10⁵).

For the measurement of ROS production, cultured podocytes were added to dichlorofluorescein diacetate (DCF-DA) at a concentration of 10 μM at 37.5°C for 30 minutes and then analyzed for fluorescence using a fluorometer (GloMax-Multi Jr Single Tube Multimode Reader, Promega Corporation, Madison, WI, USA).

The psiCHECK-2 vector (Promega, Cat #C8021) was used to construct plasmids for dual-luciferase reporter assay. RNF146 3′UTR or its mutant was cloned into the Xho І-Not I site of the psiCHECK-2 vector. Renilla luciferase activity and firefly luciferase activity were measured using GloMax-Multi Jr Single-Tube Multimode Reader (Promega) according to the manufacturer’s protocol.

Mesophyll protoplasts were isolated from four-week-old Col-0 plants according to the methods described previously (Yoo et al., 2007 ). All of the plasmids used in this assay were prepared through purification with caesium chloride/ethidium bromide (Sambrook et al., 1989 ). For the luciferase assay, protoplasts were harvested after 12-h incubation under light conditions at 23ºC with or without stimuli (50 μM ABA). The activities of LUC and GUS were measured with the GloMax-Multi Jr Single Tube Multimode Reader (Promega, USA). All experiments were repeated at least three times.

The −487 to +246 fragment of the vWF promoter was generated by DNA synthesis. The −56 ERG mutation (5′‐TTTCCTT‐3′ to 5′‐TTTAATT‐3′) and +220 GATA3 mutation (5′‐GATA‐3′ to 5′‐AACA‐3′) on the vWF promoter fragment were cloned into a pGL3‐basic vector (Promega, Madison, WT) produced by PCR. The resulting pGL3‐vWF (wild‐type, WT) and pGL3‐vWF (mutant, mut) constructs were transfected into the HUVECs with Lipofectamine 3000 (Thermo Fisher Scientific) following the manufacturer’s protocols. A plasmid containing the Renilla luciferase reporter gene was used as an internal control. After 48 h, 25 μM DHA was added to the culture and the cells were collected by passive lysis buffer after a 24‐h treatment. The cell lysates were examined using a dual‐luciferase reporter assay system (Promega) and measured by a GloMax‐Multi Jr Single Tube Multimode Reader (Promega) at wavelengths of 350–650 nm.

Luciferase Assay System (Promega, Madison, WI, USA) was used and luciferase assay was performed according to the manufacturer’s instructions. Cells infected with HIV-1 Luc⁺ virus were washed with PBS, and then lysed with lysis buffer. After centrifugation at 15,000×g for 1 min, 20 μl of sample supernatant was mixed with 100 μl of Luciferase Assay Reagent. Luciferase activity was measured in Relative Light Units (RLU) by using a GloMax®-Multi Jr Single Tube Multimode Reader (Promega, Madison, WI, USA).