Nunc lab tektm 2 chambered coverglass

All confocal imaging was performed on a Zeiss LSM 700 laser scanning confocal microscope using a 40X/1.3DIC objective. Cells were plated on 4- or 8-well Nunc^TM Lab-Tek^TM II Chambered Coverglass (Thermo Scientific) and allowed to grow for 24 h. After treatment, cells were fixed in 4% formaldehyde and imaged at excitation wavelengths of 488 nm (GFP, LysoSensor Green or BODIPY) and 560 nm (Cy3.5). ImageJ software (NIH) was used to process and analyze the images.

HeLa cervix adenocarcinoma cells (ATCC CCL-2; HeLa-wt) were obtained from ATCC (Manassas, VA, USA). The stably plitidepsin resistant HeLa cell subline (HeLa-APL-R) was generated at PharmaMar as previously described^{19 (link)}. Cells were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM, Sigma, St Louis, MO, USA) supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine and 100 units/mL of penicillin-streptomycin at 37 °C, 95% humidity and 5% CO₂. Fluorescence studies were run in Thermo Scientific^TM Nunc^TM Lab-Tek^TM II chambered coverglass (8 wells; ∼8000 cells/well; 500 μL/well) at 37 °C. Before measurements at the micro-spectrometer, cells were washed with Tyrode-glucose buffer (NaCl 145 mM, KCl 4 mM, MgCl₂ 1 mM, CaCl₂ 1.8 mM, HEPES-Na 10 mM, glucose 10 mM), pH 7.4, just before starting the measurements.
Epigallocatechin gallate (EGCG) was purchased from Sigma (St Louis, MO, USA). HeLa-wt and HeLa-APL-R cells were pre-treated with EGCG 100 μM for 30 minutes, and then loaded with different plitidepsin concentrations.