M344

MEL DS19 cells (murine erythroleukemia cells) are maintained in D-MEM containing 100 units/mL penicillin G sodium and 100 μg/mL streptomycin sulfate supplemented with 10% fetal bovine serum at 37 °C in a 5% CO₂ atmosphere. To test M344 for potential to induce cell differentiation, log-phase cells with a population doubling time of 11−13 hours are used. Serial dilutions of M344 are prepared in 24-well plates using 1 mL of D-MEM/well. If M344 are dissolved in DMSO, control wells contains the same amount of solvent (generally 2 μL/mL of medium). Subsequently, the cell suspension is added to the wells. After 72 hours the experiment is evaluated. Cell numbers are counted using a Casy 1 TTC flow cytometer. The proliferation of treated cells is expressed as percent proliferation in comparison with the solvent control. Differentiated MEL cells accumulate hemoglobin. Therefore, the induction of cell differentiation is determined by benzidine staining according to the literature. To 100 μL of cell suspension is added 10 μL of a 0.4% solution of benzidine in 12% acetic acid containing 2% H₂O₂. Within 5 minutes hemoglobin-containing cells stains blue. Benzidine-positive and -negative cells are counted under the microscope in a hemocytometer, and the percentage of positive cells is calculated. M344 is first tested at 10 μM and 50 μM final concentration. According to activity/toxicity profile, a range of concentrations is chosen for a dose−response analysis.