Synergy 2 multi mode biotek plate reader

P-gp is an ATP-dependent drug efflux pump that plays an important role in multi-drug resistance and drug bioavailability. The effect of the PEGylated vitamin E isomers on the P-gp ATPase activity was carried out by the Pgp-Glo™ assay (Promega Corporation, Madison, WI). The Pgp-Glo™ assay detects the effects of compounds on recombinant human P-gp in a cell membrane fraction. The assay relies on the ATP-dependent light-generating reaction of firefly luciferase where an increase in luminescence is indicative of the inhibitory effect of compounds on the P-gp ATPase enzyme. PEGylated vitamin E isomers at a final concentration of 10 to 100 μM in assay buffer were added to the wells of a white 96-well plate containing 20 μL (0.5 mM) verapamil, which was required to activate P-gp ATPase (Collnot et al., 2007 (link)). The reaction was initiated by adding Mg ATP (25 mM) to each well. The plate was placed on a shaker for 5 min and then incubated for 40 min at 37 °C. The reaction was stopped by adding 50 μL of the ATP detection reagent. After addition, the plate was left at room temperature for 20 min to allow for the luminescent signal to develop. Luminescence was measured using a Synergy 2 Multi-Mode BioTek plate reader (BioTek Instruments, Inc. Winooski, VT).

The susceptibility of the gemcitabine-lipid conjugates to deamination was evaluated using Human Cytidine deaminase (CDA) ELISA kit (BioVisiosn Inc., Milpitas, CA). The CDA enzymatic solution was diluted with 50 mM Tris-HCL buffer (pH 7.5) to make an enzymatic solution with a concentration of 0.25 μg/50μL. Solutions of free gemcitabine HCL (100 μM) or gemcitabine-lipid conjugates (solubilized in DMSO, 100 μM equivalent to gemcitabine HCL) were prepared in 50 mM Tris-HCL (pH 7.5). The solutions of the free gemcitabine or gemcitabine-lipid conjugates (150 μL) were pipetted into a 96-well white plate. The enzymatic solution (50 μL) was then added to each well, and the plate was placed on a shaker for 2 min. The absorbance of each well was measured at 280 nm for 60 min at 37°C using a Synergy 2 Multi-Mode BioTek plate reader (BioTek Instruments, Inc. Winooski, VT).