Xmark microplate absorbance spectrophotometer

PC3 and 22RV1 siBAT1 or BAT1cDNA cells were seeded at a density of 1x10⁴ cells/well in a 96-well plate. Cell proliferation was assayed at 24 hours using 20 µL of CellTiter 96^® AQueous One Solution Reagent (Promega, Madison, WI, USA) and 100 µL of RPMI medium, and incubated for 2 hours at 37°C and 5% CO2 in a humidified incubator. The plates were read at 490 nm using the xMarkTM Microplate Absorbance Spectrophotometer (Bio-Rad, Hercules, CA, USA).

To evaluate cytotoxicity of free IDE, the IDE/HP-β-CD inclusion complex, and overloaded and non-overloaded CS NPs, a methylthiazolyldiphenyl-tetrazolium bromide (MTT) test was carried out. The U373 cells were plated in 96-multi-well dishes (8 × 103 cells/0.1 mL) and treated with different samples containing 6.25, 12, and 25 µM of IDE. The cells were incubated for 24, 48, or 72 h, and then 10 µL of tetrazolium salts (5 mg/mL in PBS, pH 7.4) were added to the dishes and incubated for 3 h. At the end of the incubation, cell viability was calculated by an ELISA microplate reader (BIO-RAD, xMark^TMMicroplate Absorbance Spectrophotometer, Hercules, CA, USA) at λ_abs = 570 nm and λ_abs = 670 nm according to the following equation:
where AbsT/AbsC represents the ratio of cells treated (AbsT) and untreated control cells (AbsC). Three independent experiments were performed and data are reported as the mean of results ± standard deviation (S.D.).

Liver damage of OMGVac was evaluated by ALT1 and GOT 1 enzyme-linked immunosorbent assay (ELISA) kits (MyBiosource Inc., San Diego, CA, USA) according to the manufacturer’s protocols. In brief, 50 µL of undiluted hamster sera were added to the pre-coated ALT1 and GOT1 96-well plate. Then, 100 µL of HRP-conjugated reagent was added to two plates, and the plates were incubated in a 37 °C incubator for 60 min. After several washing steps, chromogen solutions A and B were added to each well, and the plates were incubated at 37 °C for 15 min. The stop solution was added to the plates, and the optical density at 450 nm was measured by an xMark^TM Microplate Absorbance Spectrophotometer (BioRad, USA).

Cell proliferation analysis of siRNA-transfected cultures was performed using TFK1 and HuCCT1 cells. These cells were cultured in 96-well culture plates, at 10,000 cells/well, in 100 μl of medium. After initial cell seeding, cell viability was analyzed using the Cell Counting Kit-8 (Dojindo Laboratories, Kumamoto, Japan). Evaluations were performed after 0, 24, 48, 72, and 96 h. The cell counting solution was added at a concentration of 10 μl/well, and cells were incubated at 37°C in a humidified 5% CO₂ atmosphere for 2 h 30 min. The optical density of the wells was measured at 450 nm using an xMarkTM Microplate Absorbance Spectrophotometer (Bio-Rad Laboratories, Hercules, CA, USA). All results were derived from 12 sets of duplicated experiments.

Alkaline phosphatase enzymatic activity was measured according to an established protocol. Briefly, cells were collected in 0.2% Triton-X and homogenized using an ice-cold Dounce grinder. The protein concentration was measured using Pierce^TM BCA protein Assay Kit (23225, Thermo Scientific). Alkaline phosphatase enzymatic activity was measured using p-Nitrophenylphosphat solution (P7998, Sigma) pH 10.5 as substrate and 4-nitrophenol solution (N7660, Sigma) diluted in 0.2% Triton-X as a standard. Plates were read at 405 nm (xMark^TM, Microplate Absorbance Spectrophotometer, 1681150 BioRad). A total of 6 wells per group were evaluated.

Prior to the experiment, OCCM-30 cells were cultivated overnight in starvation media: α-MEM (11095-080, Gibco) supplemented with 0.5% FCS (10270-106, Gibco), 1% penicillin/streptomycin (15140-122, Gibco), 50 μg/ml ascorbic acid (Art. 6288.1, Roth), and 10 mM β-glycerophosphate (#35675, Calbiochem). Cells were cultivated either under compression of 2.4 g/cm² or without compression and with and without the addition of 50 ng/ml leptin (CYT-351, Prospec). To evaluate the effect of ERK1/2 activation on cytosolic phospholipase A2 (cPLA2) regulation, the ERK inhibitor II FR180204 (328007, Calbiochem) was employed. The inhibitor FR180204 (0.2 μg/ml) was added to the cell culture 1 h before experiment start. Cells were collected in phosphate buffer (pH 5.8) and sonicated (Branson Sonifier 150). Phospholipase activity was detected using Cytosolic Phospholipase A2 Assay Kit (ab133090, Abcam) according to the manufacturer protocol. Plates were read at 405 and 414 nm (xMark^TM Microplate Absorbance Spectrophotometer, 1681150 Bio-Rad).

Cell proliferation analysis was performed using the Cell Counting Kit-8 (Dojindo Laboratories, Kumamoto, Japan). At 24 h after RAB5 siRNA transfection, the SUIT-2 cells were plated in 96-well plates in 100 µl of medium containing 10% FBS at approximately 2000 cells per well. Evaluations were performed at 0, 24, 48, and 72 h. For quantifying cell viability, 10 µl of cell counting solution was added to each well and incubated at 37°C for 2 h 30 min. Next, the absorbance of the well was detected at 450 nm using the xMark^TM Microplate Absorbance Spectrophotometer (Bio Rad, Hercules, CA, USA).