8 wells chamber slide

ER-E2F1 U2OS cells were plated in 8 wells chamber slide (Ibidi) and loaded with a 70, 000-Da dextran that was coupled to FITC and to Rhodamine (Fluorescein isothiocyanate–dextran, Rhodamine B isothiocyanate Dextran) (Sigma-Aldrich, USA) at 1 mg/ml during 20 hours in serum starved conditions. After washing, cells were treated as indicated. Fluorescence was analyzed on the Leica TCS SP5 spectral Live confocal microscope using a 63X N.A 1.4 objective and LAS AF software. Time-lapse images were taken from six regions for each condition every minute during 20 hours using excitation wavelengths of 488 for FITC and 568 for Rhodamine. The fluorescence intensity of the images was analyzed using ImageJ software. As a result, the red and green signals as a function of time were obtained. These curves featured a noisy pattern, so that each of them was smoothened by a polynomial interpolation. The green-to-red ratio was computed from each of these smoothened curves and then from these an average curve with standard deviation was computed for all six positions. Calibration was performed by incubation of the cells with media adjusted between 5.0 to 8.0 pH values containing 10 μM of nigericin (Panreac Sciences, Spain) and 10 μM Valinomycin (Sigma-Aldrich, USA).

ER-E2F1 U2OS cells were plated in 8 wells chamber slide (Ibidi) and transiently transfected for 24 h to express LAMP1-GFP. After overnight serum starvation, cells were treated as described. Fluorescence was analyzed on the Leica TCS SP5 spectral Live confocal microscope using a 63X N.A 1.4 objective and LAS AF software, within an incubation chamber XL LSM710 S1 (PeCon GmbH, Germany) with a heating insert P-LabTek S1. GFP fluorophore was excited with Argon laser 15%. Time-lapse images were taken from six regions for each sample every minute for 20 hours. Images were analyzed with ImageJ software and converted into avi format to be edited with Final Cut software.