Resazurin assay

LC-2/ad cells obtained from ATCC were cultured in a humidified atmosphere with 5% CO₂ at 37 °C using the recommended 1:1 ratio of RPMI-1640 (Gibco, Carlsbad, CA) to F12K (Corning, Corning, AZ) growth medium supplemented with 10% FBS (Gibco, Carlsbad, CA), 100 U/mL penicillin (Gibco, Carlsbad, CA), and 100 U/mL streptomycin (Gibco, CA, UCA). Cell viability assays were performed in 96-well plates in triplicate by exposing cells to seven concentrations of the studied compounds for 72 h. Then, a resazurin assay (Biotium, Fremont, CA) was used according to manufacturer instructions. Fluorescence was monitored at 540 (excitation) and 590 nm (emission) using a Synergy Neo2 microplate reader (Biotek, Winooski, VT). GI₅₀ values were calculated from the dose–response curves, which were generated in GraphPad Prism 8 (GraphPad Software, San Diego, CA. Error was determined as the standard deviation between two independent measurements.

The FLT3 mutants driven MOLM14 cell lines and Ba/F3 cell line were obtained from the laboratory of Dr. Neil Shah (University of California, San Francisco). All the cells were cultured in a humidified atmosphere with 5% CO₂ at 37 °C. RPMI-1640 (Gibco, Carlsbad, USA) medium with 10% fetal bovine serum (FBS) (Gibco, Carlsbad, USA) were used to culture MOLM14 cell lines. While Ba/F3 cell line used the same growth medium as MOLM14 but with additional 2 ng/ml murine IL3. Cells were treated with compounds (200μM–0.0002 μM) and proliferation was assessed using resazurin assay (Biotium, Fremont, USA) following the manufacturer’s instruction. The plates were read at 540 nm (excitation)/590 nm (emission) using a Synergy Neo2 microplate reader (Biotek, Winooski, VT). Then, IC₅₀ values were calculated from the dose-response curves which were generated in GraphPad Prism 8 (GraphPad Software, San Diego, USA). Error was determined as the standard deviation between three independent IC₅₀ measurements.