Nastrp

Cells were seeded in 96 well plates at 2×10³ cells/well with RPMI-1640 medium supplemented with 5% heat-inactivated FBS and without Anti-Anti. Cells were treated with Naphthol AS-TR phosphate disodium salts (NASTRp, Sigma-Aldrich, N6125) as 0–80 μmol/L for 96 hours. Cell proliferation was measured with MTT or CellTiter Glo luminescent cell viability assay (Promega, Madison, WI, USA). Cell viability of vehicle-treated cells was set to 100% of proliferation. For colony formation assay, cells were seeded in 6 well plates at 1×10³ cells/well. Cells were treated with NASTRp as 0, 5, 10, and 20 μmol/L and were changed with fresh media containing NASTRp every other day for 10–17 days at which point 0.1% (wt/vol) crystal violet (Thermo Scientific, NJ, USA) was used to visualize colonies. Soft agar assay was performed as described previously [12 (link)]. Briefly, colonies were stained with p-iodonitrotetrazolium violet (Sigma-Aldrich) to select alive colonies and then, stained colonies were counted using Image J software (NIH, USA). A colony was defined as anything containing more than 10 cells, as indicated >50 pixels in Image J.