Doxorubicin Uptake Assay

Intracellular doxorubicin concentrations were measured with or without TRPV2 activators. BNL1 ME cells were plated on six-well plates at concentrations of 2 × 10⁵ cells/well and treated with DMEM for 24 h, after incubation at 37°C for 1 h. With each one of the treatments, each culture medium was removed, and cells were washed three times with PBS. The cells were lysed in 1 ml HCL-acidified isopropanol for 24 h (centrifuged at 2,000 rpm for 10 min). For fluorometric analysis, total cellular doxorubicin in BNL1 ME cells was determined by measuring the fluorescent emission of the solution (λ_ex = 480 nm, λ_em = 590 nm) in the cell lysate with a fluorometer, as described elsewhere (Patil et al., 2018). The drug concentration was calculated with the standard curve of doxorubicin.