Mtt cell proliferation assay

The effect of drug on cell proliferation was determined by using TACS 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) cell proliferation assay (Trevigen, Gaithersburg). The cells (2,000 per well) were incubated with or without morphine in triplicate in a 96-well plate and then incubated for 2, 4, and 6 days at 37°C. A MTT solution was added to each well and incubated for 2 h at 37°C. An extraction buffer (20% SDS and 50% dimethylformamide) was added, and the cells were incubated overnight at 37°C. The absorbance of the cell suspension was measured at 570 nm using a microplate reader (DAS Technologies, Chantilly, VA). This experiment was repeated twice, and the statistical analysis was performed to obtain the final values.

Cell viability was evaluated using the MTT Cell Proliferation Assay (Trevigen, Gaithersburg, USA). The yellow tetrazolium salt is intracellularly reduced to purple formazan. After cell-lysis the amount of formazan can be measured. As analytical parameter for loss of cell integrity the cytosolic lactate dehydrogenase was chosen. The enzyme activity in cell free supernatants was measured with the Cytotoxicity Detection Assay (LDH, Roche). All assays were performed according to the manufacturers’ instructions and absorptions were measured with an ELISA-Reader.

Cell proliferation was measured by MTT cell proliferation assay (Trevigen, Gaithersburg, USA). Following transfection cells were plated (3×10³ cells/well) in 96-well plates with no phenol red RPMI medium mixed with 10% fetal bovine serum and then cultured at 37°C for 24 h. Then CXCL10 and CXCL11 were added at 100 ng/mL and the cells were cultured for 48 h and 10 μl of MTT reagent was added to each well. When purple crystals of formazan became visible under the microscope, 100 μl of detergent reagent was added, and the cells were incubated for 2 h. Absorbance of cells in each well was observed at 570 nm under an absorption spectrophotometer (Autobio Labtec, China) and corrected against blanks (culture medium). Cells transfected with empty vector (pTopo) were considered as control. All experiments were conducted independently for 3 times. The reading at 570 nm is directly proportional to cell proliferation (number of viable cells).