Glomax microplate reader

Mouse Neuro2a (N2a) cells (CCL-131; American Type Culture Collection, Manassas, VA) were plated at 80,000 cells/well in 24-well tissue culture dishes and, 24 h later, were cotransfected with 100 ng miR-29 target reporter plasmid plus 10 nM either control miR-Neg or miR-29a pre-miRNA synthetic oligos (Ambion, Austin, TX), along with 100 ng either TuD-Ctrl plasmid or TuD-29 expression plasmids, using Lipofectamine 2000 (Thermo Fisher Scientific, Waltham, MA), completed in triplicate for each treatment combination. At 48 h posttransfection, FF and R luciferase activities were measured using a GloMax Microplate Reader and Dual Luciferase Kit reagents (Promega). Briefly, culture media was removed and 200 μL Passive Lysis Buffer was pipetted into each well, and the plate placed on a shaker for 15 min at room temperature. Then, 10-μL lysate was transferred to duplicate wells of a 96-well white plate. Luminescence from FF and R was determined from 5-s reads after the injection of respective substrates to each well. The R/FF ratio was calculated and adjusted relative to the control (set to “1”), and results are expressed as the mean ± SEM (n = 4/group).

Cell viability was evaluated using the calorimetric MTT assay. Each experiment was performed in triplicate. Cells were seeded in 96-well plates at (8 × 10³ cells per well) in the case of HDF and at (9 × 10³ cells per well) in the cases of MCF7 and MDA-MB-231 and cultured for 24 h. After which, the cells were incubated with free TC, blank CS-SP NPs, or TC-CS-SP NPs at different concentrations at 37 °C and once again at 40 °C (5 min per 24 h).^{70 (link)} In this instance, the elevated temperature was adjusted by raising the temperature of the incubator. For the negative control, untreated cells were incubated with the complete culture medium. After 72 h of incubation at 37 °C, treatments were removed from the wells, followed by adding 15 μL of 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium bromide (MTT) solution and 100 μL of the medium to each well, and the cells were incubated at 37 °C for further 3 h. Then, the medium and MTT salt were removed and replaced with 50 μL of dimethyl sulfoxide (DMSO) to dissolve the insoluble formazan. Absorbance values were measured at a wavelength of 570 nm using Glomax microplate reader (Promega, USA). Results on relative cell viability were presented as the percentage of surviving cells in relation to untreated cells.