ADI-based Autophagy Induction in Prostate Cancer Cells: An Enzyme-based Technique to Measure Autophagic Response in Cells

1. Preparing Cells for Live Imaging

1. Grow CWR22Rv1 cells expressing green fluorescent protein-coupled Light Chain 3 (LC3-GFP) in RPMI culture media containing 10% FBS and 1% antibiotics on 35 mm poly-D-lysine coated glass-bottom culture dishes. 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.
2. Treat selected cell samples with ADI (0.3 μg/mL) in PBS.
3. Approximately 1 hr prior to imaging, dilute 1.5 μl of LysoTracker Red DND-99 (Invitrogen, CA) with 20 mL RPMI containing 10% FBS and 1% antibiotics. Use solutions containing ADI for selected samples. Warm all media to 37 °C prior to adding it into the culture dish.
4. Incubate cells with RPMI containing LysoTracker Red DND-99 for 15-45 min at 37 °C.
5. Approximately 30 min prior to imaging, turn on the WeatherStation environmental enclosure and allow to equilibrate to 37 °C and 5% CO₂ (with humidified air).
6. Wash cells with PBS and replace media with standard RPMI containing only 10% FBS and 1% antibiotics. Add ADI to samples as indicated.
7. Mount 35 mm coverglass-bottom culture dishes in customized adaptor (Applied Precision, Inc., WA), and position on the microscope stage. Use immersion oil (refractive index 1.520) on the 60X 1.42 NA objective lens and position the mounted culture dish on the stage.

 2. Super-resolution, Structured-illumination (OMX) Microscopy

NOTE: The protocol in this section applies to the use of the OMX Structured Illumination Microscope (Applied Precision, WA).

1. Switch on the main power and desired lasers (410 nm, 488 nm, and/or 532 nm). Wait for 20 min for the lasers to be thermally stabilized.
2. Add immersion oil (refractive index 1.516 for fixed samples at RT) on the 60X 1.42 NA objective lens. If bubbles are seen in the oil droplet, clean the objective and add oil again.
3. Place sample slide on the stage and if necessary, allow 10 min for cells to settle on coverslip.
4. Initialize the acquisition program. Use fluorescence illumination to adjust the focus until a sharp outline of the cell can be seen.
   NOTE: Care should be taken at all times to minimize exposure of cells to fluorescence excitation, until the actual image acquisition.
5. A spiral mosaic scan can be acquired to preview larger areas of the sample to select cells or regions of interest. It is recommended to use short exposure times (1-10 msec) and low excitation power (0.1-1% transmission) while scanning the sample or finding targets.
6. Identify autophagosomes by GFP-LC3 fluorescence. Identify lysosomes by Alexa Fluor 555 anti-LAMP (lysosome-associated membrane protein) and cell nuclei using DAPI (fixed samples).
7. Choose experimental conditions including excitation wavelength, image area (e.g., 512×512), stack thickness, exposure time, and laser transmission percentage. For super-resolved images, ensure that the structured illumination option is enabled. For deconvolution microscopy on the OMX, select the conventional illumination option.
8. For the best reconstruction results, select experimental conditions such that the maximum intensity count is between 10,000 and 15,000 during the acquisition. If photobleaching is a concern, imaging at a lower exposure (counts between 5,000 and 10,000) may be used. Ideally, the counts should not decrease more than a factor of two during the entire acquisition, and decreases by more than a factor of four should be avoided. If necessary, reduce the stack thickness to ensure a sustained intensity count. The camera saturates at 64,000 counts, so maximum intensity should never exceed this. For good samples, we found the experimental conditions to be 10~50 msec exposure time with 1% laser transmission. Bright and photostable samples that can be imaged repeatedly are preferred. A stable stain is required for time-lapse, super-resolved images.
9. To reduce noise, a 95MHz readout speed (Medium speed option) and acquisition time of 2 msec or more is recommended. Increased artifacts are obtained in the reconstructed image when the sample moves during the acquisition. Due to this effect, short exposures are recommended for non-adherent or fast-moving cells.
10. Multichannel imaging can be performed simultaneously or sequentially. Sequential mode yields less crosstalk and is thus recommended.
11. To reduce photobleaching, it is generally recommended to image with longer excitation wavelengths first (532 nm, 488 nm, and then 410 nm). However, for the experimental results shown in Figure 1, this order was reversed (i.e., 48 nm, 532 nm and 410 nm) due to the photostability of this particular sample.
12. Set the upper/lower limit of the Z-stack by moving the microscope stage until the top/bottom of the cells is slightly out of focus. The desired distance between each image should be held constant at 0.125 μm for super-resolution imaging, as reconstruction software takes this value as default.
13. Reconstruct image stacks using SoftWorX (Applied Precision, WA) and analyze using VoloCITY 6.0 (Perkin Elmer, MA).