mts test cell titer 96 aqueous one-solution cell proliferation assay  (Promega)

Journal: Pharmaceuticals

Article Title: Combination Treatment of Resistant Acute Promyelocytic Leukemia Cells with Arsenic Trioxide and Anti-Apoptotic Gene Inhibitors

doi: 10.3390/ph17111529

Figure Lengend Snippet: SMAC mimetics’ growth inhibitory effect is independent of ATO resistance and IAPs RNA expression. ATO-resistant clones were treated with the indicated concentrations of LCL161 ( A ) or xevinapant ( B ). After three days, the proliferation was evaluated by cell count in triplicate. Data are represented as surviving fractions and are the mean ± SD of three independent experiments. The IC 50 for each cell clone is indicated on the right of each graph. NE = not evaluable. ( C ) RNA expression of BIRC3 , BIRC2 , and XIAP was measured by RT-PCR using the total RNA extracted from cultured cell clones. Relative expression was obtained by normalizing to GAPDH and calibrating to the NB4 bulk cell line. Data are presented as the mean ± SD of triplicate RNA extractions. ( D ) CL1 and CL1-R cells were treated for three days with the indicated concentrations of ATO (left panel) or LCL-161 (right panel). Data are represented as surviving fractions and are the mean ± SD of three independent experiments evaluated by cell count. ( E ) IC 50 mean values are shown. ( F ) ATO-resistant clones were treated with 1 µM of ATO plus graded concentrations of LCL161 (0.25–16 µM). The combined effects were evaluated in the four ATO-resistant clones after 48 h of drug treatment by MTS test and analyzed using CompuSyn software. Each Fa-CI plot (or Chou–Talalay plot) indicates the CI as a function of the fraction affected (Fa). CI < 1, synergistic (values below the dotted line); CI = 1, additive; CI > 1, antagonist. Statistical analysis was performed using the unpaired Student’s t -test: * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: For the survival assays, the cells were analyzed by the MTS metabolic test (CellTiter 96 Aqueous One Solution Cell Proliferation Assay, Promega, Madison, WI, USA), according to the manufacturer’s instructions, or by the cell count viability test with trypan blue dye.

Techniques: RNA Expression, Clone Assay, Cell Counting, Reverse Transcription Polymerase Chain Reaction, Cell Culture, Expressing, Software