Xl lsm 710 s1

A custom designed construct was used to grow bacterial colonies under the microscope as previously described (Mamou et al., 2016 (link)). Bacterial cells were spotted at a concentration of ~1 cell/ μl on solid LB medium. When indicated, PI (Sigma) was added to a final concentration of 5 μg/ml. Construct was covered with a 35 mm cultFoil membrane (Pecon) to reduce dehydration, and incubated at 37°C in Lab-Tek S1 heating insert (Pecon) placed inside an incubator XL-LSM 710 S1 (Pecon). Developing colonies were visualized and photographed by CLSM LSM700 (Zeiss) with Plan-Aporchromat x10/NA0.45 (Zeiss). Cells expressing GFP or Dronpa were irradiated using 488 nm laser beam, while cells stained with PI were irradiated using 555 nm laser beam. For each experiment, both transmitted and reflected light were collected. System control and image processing were carried out using Zen software version 8.0 (Zeiss).

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.