Biotek microplate reader

Microemulsion penetration was investigated using Franz-type diffusion cells (PermeGear Inc., Hellertown, PA, USA) with a diffusion area of 1 cm² and an acceptor compartment of 8 mL containing fetal bovine serum and PBS (pH 7.4) (1 : 9, v/v). Skin was mounted on Franz-type diffusion cells, epidermal side up, and dermal side facing the receptor compartment. Diffusion cells were kept at 32°C. 100 μL of different treatments (the final curcumin concentration was 1%w/w (27.1 mM)) was applied to the mounted skin. Following 24 h incubation, skin was removed and washed three times using a cotton cloth containing ethanol and the viable epidermis was separated from the dermis. Separation of the full epidermis from the dermis was achieved by heat shock treatment; skin was placed for 30 seconds at 55–60°C followed by 1 min at 4°C, both in PBS. Curcumin was extracted from the separated layers with DMSO. The extraction was performed by incubation in a shaker (60 ×g) until all curcumin were released (24 h). Finally, 100 μL from the receptor fluids was collected. Curcumin existence in skin layers and in the acceptor compartment was determined by measuring fluorescence excitation at 485/40 nm and emission at 528/20 nm, using a BioTek microplate reader (BioTek Instruments Inc., Winooski, VT).

A549 (2 × 10³) cells were seeded in 80 µL of DMEM supplemented with 10% FBS in a 96-well plate. After overnight incubation, 80 µL of media containing either 0.01% (v/v) DMSO or 1 µM Barasertib (AURKB inhibitor) or 5 µM BI-D1870 (RSK inhibitor) was added to the cells. In parallel, cells were also transfected with 25 ng of either Vo or YB-1:EBFP2 using Lipofectamine 3000 (LF3000, Invitrogen) as described above. Each treatment had six biological replicates. Twenty microliters of the lipoplexes were added to the cells. At the indicated time points the medium was removed and the plates were frozen at –80 °C. After all the time points had been collected the plates were thawed and the DNA content measured using a SYBR Green I-based fluorimetric assay as described previously [74 (link)]. Briefly, 100 µL of lysis buffer (10 mM Tris-HCl pH 8.0, 2.5 mM EDTA and 1% (v/v) Triton X-100) containing 1:4000 SYBR Green I (Invitrogen) was added to the wells and the plates were incubated overnight at 4 °C. The plates were mixed and the fluorescence signal for each well was measured for 1 s at an excitation of 485 nm and emission of 535 nm using a BioTek microplate reader (BioTek, Winooski, VT, USA). Growth curves were plotted as the fluorescence values at each time point. There were at least three biological replicates for the control and the treated samples.

Bulge cells were cultured and treated as described above. The cell proliferation assay was performed using CellTiter 96^® AQueous One Solution Cell Proliferation Assay (MTS) Kit (Promega) following the manufacturer’s instructions. Absorbance at 490 nm was examined in a BioTek Microplate Reader (BioTek Instruments Inc.). For statistical analysis, every group had a triplicate. Data were normalized to the control group. Data are presented as means ± SEM. and differences were considered significant with a p value less than 0.05.