ADI-based Autophagy Induction in Prostate Cancer Cells: An Enzyme-based Technique to Measure Autophagic Response in Cells
내레이션 대본
To begin, grow human prostate cancer cells expressing green fluorescent protein-coupled Light Chain 3 on 35-millimeter poly-D-lysine coated glass bottom culture dishes, as described in the text protocol.
Cells should be plated at sufficient density to facilitate rapid proliferation, but not so much that cells are overgrown and clumped by the time of imaging. Treat selected cell samples with arginine deaminase, or ADI, in PBS to deplete cells of free arginine and induce metabolic stress in the cancer cells.
Approximately one hour prior to imaging, dilute 1.5 microliters of LysoTracker Red with 20 milliliters of RPMI containing 10% FBS and 1% antibiotics. Prepare solutions with ADI for the selected samples that were treated.
After warming all media to 37 degrees Celsius, add the appropriate media to each culture dish. Incubate cells with RPMI containing LysoTracker Red for 15 to 45 minutes at 37 degrees Celsius.
Approximately 30 minutes prior to imaging, turn on the weather station environmental enclosure and allow equilibration to 37 degrees Celsius and 5% carbon dioxide. Wash cells with PBS and replace media with standard RPMI containing only 10% FBS and 1% antibiotics.
Add ADI to samples as indicated. Mount 35-millimeter cover glass bottom culture dishes in a customized adapter. Use immersion oil on the 60X magnification, 1.42 numerical aperture objective lens, and position the mounted culture dish on the microscope stage.
