Veritas microplate luminometer

293T cells were seeded in a 96-well plate, cultured to 70% confluence, and transfected with either the h-mTOR-3′ UTR plasmid or hsa-miR-100-5p/Negative Control (Hanbio Biotechnology, China) using a transfection reagent (Hanbio Biotechnology, China). The cells were collected at 48 h after transfection. Luciferase activity was determined in cell lysates with a Dual-Luciferase Reporter Assay System (Promega, USA) according to the manufacturer’s instructions. Firefly luciferase and Renilla luciferase activities were detected on a Veritas Microplate Luminometer (Promega, USA). The firefly luciferase to Renilla luciferase ratio was calculated for each sample and was normalized to the ratio for NG-cultured cells.

The xopAU, xopAU_K240A, MKK2, MPK1, BSK830 (GenBank acc. num. XP_004252882.1) and BTI9 [78 (link)] genes were cloned in frame to firefly luciferase fragments in the binary vectors pCAMBIA:N-LUC and pCAMBIA:C-LUC [79 (link)]. The obtained vectors were transformed into Agrobacterium and co-expressed in N. benthamiana leaves. Luciferase activity was measured at 48 h after infiltration: 3 mm diameter leaf disks were harvested and floated in 100 μL water in a white 96-well plate. Samples were supplemented with 0.5 mM D-luciferin (Sigma-Aldrich, St. Louis MO, USA) and incubated in the dark for 10 min. Luminescence was measured using a Veritas Microplate Luminometer (Promega Corporation, Madison WI, USA).

Luciferase activity in A549-GFP-luciferase cells was assessed using the Promega Luciferase Assay System according to the manufacturer’s instructions (Promega). Washed cells were digested with 40 µL of 0.25% trypsin at 37°C for 5 min. d-Luciferin substrate was diluted with RPMI-1640 containing 10% FBS at a concentration of 150 µg/mL. The digested cells were resuspended with 100 µL of d-Luciferin-medium mix. A 100-µL volume of cell suspension was transferred to a 96-well white plate. Photon emission was measured with a Veritas™ Microplate Luminometer (Promega).

To evaluate the stability and safety of Y-27632 released from the PLGA/PLA microspheres, the effect of released Y-27632 on cell growth of CECs was evaluated. Y-27632 incorporating PLGA microspheres (0020) were incubated in PBS, and PBS was recovered at 3 and 7 days after incubation. The concentration of Y-27632 was then evaluated by HPLC, and the recovered PBS including the released Y-27632 was added to the culture medium at a final concentration of Y-27632 of 10 μM. Cultured monkey CECs were seeded at a density of 5.0 × 10³ cells/cm² per well on a 96-well plate for 24 hours and then subjected to serum starvation for an additional 24 hours in the presence or absence of fresh Y-27632 (10 μM) or Y-27632 released from PLGA microspheres after 3 or 7 days (10 μM). The number of viable cells was determined by use of the CellTiter-Glo® Luminescent Cell Viability Assay (Promega, Fitchburg, Wisconsin) performed in accordance with the manufacturer's protocol. The number of CECs at 24 hours after treatment with Y-27632 was measured using the Veritas™ Microplate Luminometer (Promega, Fitchburg, Wisconsin).