Fluostar galaxy fluorimeter

CT26, B16F10 and 4T1 cells were seeded at 2 × 10⁴ cells per well in 96 well plates and grown to sub-confluency. Cells were infected at varying MOI with AdEmpty or AdFAST in a 25 μl volume for 1 h, followed by addition of 100 μl of 5% FBS/DMEM. For all experiments, the VSVΔ51 virus^{32 (link)} and cells lysed using 9% Triton X-100 for 45 min before starting the assay were used as positive controls. Each treatment condition was conducted in triplicate. Seventy-two hours post infection, 50 μl from each well was transferred to a new 96 well plate. To examine membrane permeability, the CytoTOX-ONE Homogeneous Membrane Integrity Assay kit (Promega, Madison, WI, USA) was used according to the manufacturer's instructions. Fluorescence readings were obtained using a 544 nm excitation and 590 nm emission filter on the FluoSTAR Galaxy fluorimeter (BMG Labtech, Guelph, ON, Canada).

A549 cells were seeded at a density of 2x10⁴ cells per well in 96 well plates and grown to confluency. Medium was removed and cells were infected with AdEmpty or AdFAST at a MOI of 1, 5, 10, 50 or 100 in 25 μL inoculum for 1 h. The VSVΔ51 virus [16 (link)] was used as a positive control. Medium was added to a final volume of 125 μL and the cells were incubated at 37°C in 5% CO₂ for 72 h. Prior to collecting the supernatants, a series of wells containing uninfected cells were lysed with 9% Triton X-100 to provide a maximum release control. All treatments were completed in triplicate. Fifty μL of supernatant was transferred to a new 96 well plate and the CytoTOX-ONE™ Homogeneous Membrane Integrity Assay kit (Promega) was used to assess membrane integrity. Fluorescence was determined using the 544 nm excitation filter and 590 nm emission filter with a FluoSTAR Galaxy fluorimeter (BMG Labtech).

The intracellular accumulation of Rho123 was measured using a FLUOstar Galaxy fluorimeter (BMG LABTECH, Ortenberg, Germany). The reading parameters for Rho123 were λmax = 507 nm excitation and λmax = 529 nm emission. The inhibition of the Hco-PGP-9.1 transport function by the MLs was compared against the maximum effect produced by valspodar [11 (link)] and expressed as percent of total VSP inhibition. Each experiment was repeated using three biological replicates and each drug concentration was run in triplicate. The data generated were fitted using GraphPad Prism software (Version 6).

Normal human keratinocytes were grown in a multi-96 well black plate and treated with FasL (50 ng/ml). The rate of caspase-3 and -7 activation was measured simultaneously with the fluorimetric Apo-ONE Homogeneous Caspase-3/7 Assay (Promega Corporation, Madison, WI, USA), according to the manufacturer’s instructions. Briefly, Apo-ONE Caspase-3/7 reagent mixture, containing the profluorescent substrate Z-DEVD-Rhodamine 110 and lysis/permeabilization buffer, was added to each well, gently mixed, and incubated in the dark for 2 h at room temperature. After incubation, the amount of fluorescent product, which is proportional to the amount of caspase-3/7 cleavage, was measured with a FLUOstar Galaxy fluorimeter (BMG Labtech, Germany), using an excitation wavelength of 499 nm and an emission of 521 nm. Living cell number was quantified by MTT assay in a duplicate transparent plate, by incubating in MTT solution (Sigma) for 4 h at 37°C. The formazan dye produced after DMSO solubilization was evaluated by a multiwell scanning spectrophotometer at 540 nm. The final RFLU/O.D. caspase assay results were normalized in relation to MTT values from the same experiment.