Doxorubicin

First, 100 ± 10 MCF-7 cells were loaded into the cell chip and cultured for 24 h before treatment. Doxorubicin (TargetMol, Boston, MA, USA) solutions with a concentration gradient ranging from 0, 0.462, 4.62, 11.55, to 20.79 μM were dispensed into the cell chip using the NanoCLD, resulting in final concentrations of 0, 0.2, 2, 5, and 9 μM, respectively. After 24 h of incubation, the cell chip was gently washed three times with phosphate-buffered saline (PBS) and stained with calcein AM (green) and PI (red) for 20 min. Finally, the cell chip was submerged in a fresh culture medium within a Petri dish and was photographed by the IX83 microscope. The cell viability was calculated by manually counting the numbers of live cells (green) to the total cells (green and red) and plotted with the Doxorubicin concentrations.

The transfected cells (1 × 10⁵) were seeded in 6-well plates containing uncoated cover glasses in a final volume of 2 mL. Forty-eight hours later, the culture medium was supplemented with doxorubicin (Cat No. T1020, TargetMol) at a final concentration of 10 µM, and the incubation was continued for a further 17 h. Next, the cells were fixed with 4% paraformaldehyde (Cat. No. 6148, Sigma-Aldrich) in PBS, as already described [31 (link)], followed by permeabilization with 0.25% Triton X-100 (Cat No. X-100, Sigma-Aldrich) in deionized water for 3 min. After washing with PBS, the cells were blocked with 2% BSA (Cat No. 7906, Sigma-Aldrich) in TBST for 1 h and stained with phalloidin conjugated with FITC (2 µg/mL in PBS; Cat No. P5282-.1MG, Sigma-Aldrich) and 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI; 0.4 µg/mL in deionized water; Cat No. P5282-.1MG, Sigma-Aldrich) for 30 min and 2 min, respectively. After a final wash with PBS and mounting using Fluorescence Mounting Medium (Cat No. S3023, Dako), the cells were examined using the Zeiss LSM800 confocal unit supplied with a plan-apochromatic 63x/1.4 oil DIC M27 lens (Carl Zeiss AG), as previously described [31 (link)].