Axiovision le module fluorescence lite

Cells at the log and stationary phases were harvested and examined with fluorescence microscopy to image the cellular distribution of recombinant GFP (green fluorescent protein) protein. Fluorescence was visualized on a microscope (AxioScope A1; Zeiss) fitted with a Plan Apochromat 100 × 1.40 NA DIC objective and a digital microscopy camera (AxioCam MRm Rev. 3) controlled with AxioVision LE module Fluorescence Lite (Zeiss). Images were prepared using Photoshop (version 7.0; Adobe).

All microscopy was performed using AxioVision LE Module Fluorescence Lite (Zeiss #410130-0504-000). All western blots, microscopy and clonogenic plates were processed using Adobe Photoshop CS5.1 to adjust brightness, contrast, to crop, and to convert all images to greyscale. Figures were constructed using Microsoft PowerPoint:Mac 2011 (version 14.3.6) and Adobe Illustrator (version CS5.1). All statistical calculations and graphs were made using either Microsoft Excel:Mac 2011 version 14.3.6 or Graphpad Prism (version 7.0b). All pKa calculations were carried out using ADMET Predictor version 7.2 (Simulations Plus Inc). All propidium iodide flow cytometry was processed using BD CellQuest Pro software (version 5.1). All lysotracker flow cytometry obtained on the Imagestream X Mk II were analysed using the IDEAS software (version 6.2.183).

In the immunofluorescence assays, the cells were cultured overnight in the Millicell slide (Millipore) and then exposed to different concentrations of thallium for 24 h. Cells were fixed in methanol for 20 min and acetone for 1 min. After blocking in 5% bovine serum albumin for 30 min, the cells were incubated with primary antibody diluted in a blocking solution for overnight. After washing twice with phosphate buffer saline (PBS) with Tween 20, the fluorophore-conjugated secondary antibody was added and incubated for 1 h. After washing, the samples were mounted with a mounting solution. RpL23a-GFP was amplified with forward (ATGAGATCTATGGCGCCGAAAGCGAAG) and reverse primers (GCAGAATTCTTAGATGATCCCAATTTTGTTG) and constructed in pEGFP-C1. The RpL23a-GFP was transfected with TransIT-X2 (Mirus). To detect the reactive oxygen species (ROS), the cells were stained with 2ʹ,7ʹ-dichlorofluorescin diacetate (DCFDA) (Sigma) or MitoSOX^TM (Thermo). Fluorescence was visualized on a microscope (AxioScope A1; Zeiss) fitted with a digital microscopy camera (AxioCam MRm Rev. 3), which was controlled with an AxioVision LE module Fluorescence Lite (Zeiss). The images were prepared using Photoshop (version CS3; Adobe).