br Flow cytometric detection of calcein AM accumulation in
2.7.2. Flow cytometric detection of calcein-AM accumulation in MRP1-overexpressing Flp-In HEK293 Control and MRP1-expressing Flp-In™-293 Gilteritinib were seeded into 96-well microplates. After an overnight incubation, the medium was changed, and cells were incubated for 30 min with the compounds in the presence of 0.2 μM calcein AM, a non-fluorescent compound that turns into Toxicology and Applied Pharmacology 362 (2019) 136–149
fluorescent calcein inside live cells and is then further transported by ABCC1 (Baiceanu et al., 2016). The cells were then washed with ice cold PBS and stored on ice until measurement. Calcein fluorescence was de-tected on a Macsquant VRB (Miltenyi Biotec) flow cytometer, using the blue laser for excitation (488 nm) and emission in the 525/50 nm window.
2.7.3. Flow cytometric detection of mitoxantrone accumulation in BCRP-overexpressing HEK293 cells
Control and BCRP-expressing HEK293 cells were seeded into 96-well microplates. After an overnight incubation, the medium was changed, and cells were incubated for 30 min with the compounds in the presence of 5 μM mitoxantrone and then washed with ice cold PBS and stored on ice until measurement. Compounds were initially tested at 20 μM and further tested at 10 and 1 μM for those giving > 80% inhibition at 20 μM. Mitoxantrone fluorescence was detected on a Macsquant VRB (Miltenyi Biotec) flow cytometer, using the red laser for excitation (635 nm) and emission in the 655–730 nm window.
2.7.4. Compound inhibition efficacy evaluation
The efficacy of compounds in inhibition of calcein and mitoxantrone efflux was estimated by using Eq. (1),
where FA and FBG (background) correspond to the intracellular fluor-escence of the cells incubated with (FA) or without (FBG) fluorescent substrate, in the presence of each tested compound, Fe corresponds to FA measured on control non-transfected cells. Assays were performed in triplicate.
2.8. Measurement of doxorubicin resistance reversal in MES-SA/DX5 cells
MES-SA/DX5 cells were seeded into 96-well plates with a density of 4 × 104 cells/ml. After incubation for 24 h at 37 °C, cells were treated with two or three different concentrations of synthesized compounds (1, 5 and 25 μM) in triplicate and incubated for 1.5 h at 37 °C. Then doxorubicin was added at three different concentrations and the cells were further in-cubated for 48 h. The final concentrations used in this test were 0.01, 0.3, 0.1, 0.3, 1, 3 and 10 μM. Depending on the activity of the tested compound in reducing the IC50 of doxorubicin, three different concentrations were chosen among these concentrations. Afterward, 80 μl of MTT solution (5 mg/ml) in phosphate-buffered saline was added to each well for 4 h, followed by addition of 200 μl DMSO. Finally, absorbance was detected at a wavelength of 570 nm with background correction at 650 nm using a microplate reader (model 680, Bio-Rad, Japan) and the concentrations that results in 50% inhibition of cell viability (IC50s) were calculated with Curve Expert version 1.34 for windows.
2.9. Measurement of cell viability of BHK-21 and MRP1-transfected BHK-
The sensitivity of control BHK-21 and MRP1-transfected BHK-21 to different compounds was determined using a standard MTT colori-metric assay. Briefly, cells were seeded into 96-well plates at a density of 2 × 103 cells/well in 100 μl of medium. Cells were cultured over-night and 100 μl of fresh medium containing various concentrations of tested compounds dissolved in DMSO (0.5–100 μM) were added to each well. DMSO final concentration was equal to or lower than 0.5%. After 72 h of incubation, MTT assay was performed as described earlier. The effect of test compounds on cell viability was determined from the difference in absorbance between test wells versus solvent control wells. IC50 was calculated from the obtained dose–response curves of concentrations of tested compounds against plotted percentages of cell viability. Selectivity ratio (SR) was calculated as the ratio between IC50 for control cells and IC50 for MRP1-transfected cells.