長期的な住セルイメージ投射細胞の運命をパクリタ キセルに応答を評価するために

The overall goal of this procedure is to use live-cell imaging to assess cell fate decisions following treatment with the anti-mitotic drug paclitaxel. This method enables us to visualize how individual cells respond to different doses of the anti-mitotic drug paclitaxel. The main advantage of this technique is that it provides a wealth of information on single cells or whole populations that is unobtainable from fixed-cell imaging or population-based assays.

Demonstrating this method is Marc Vittoria, a MD/PhD candidate in the Ganem Laboratory. To begin, clean the glass bottom of the imaging dish with optical cleaner to remove any fingerprints or dust that may interfere with imaging. Use sufficient optical cleaner to wet the entire glass surface.

Place the glass-bottom dish with cells on the microscope stage in the imaging dish adaptor. Remove the plastic cover from the dish, and cover the dish with a glass-topped chamber. Ensure the chamber has a valve to allow a steady flow of air consisting of 5%carbon dioxide.

To humidify the 5%carbon dioxide, the gas is flowed through tubing inserted into a sterile water bath housing. This allows for the gas to become equilibrated to 95%humidity. Initiate and calibrate the encoded stage using the software program controlling the microscope.

This will ensure accurate XY coordinates and prevent focal drift. Next, perform the Kohler illumination by raising the condenser and closing the aperture so that the field is dark. Lower the condenser until a crisp hexagonal shape of light is visible.

Then, use the centering screws to center the hexagon of light in the field of view. Once the hexagonal shape is visible, open the aperture until the whole field of view is illuminated. Engage the microscope's auto-focus to ensure that all points are maintained in focus through the duration of the experiment.

Use the acquisition software to select several non-overlapping fields of view from each well for imaging. Select imaging regions where cells have adhered well to the glass bottom and are between 50 and 70%confluent. Avoid areas of clumped cells, as this will make subsequent analysis difficult.

To assess mitotic cell fate, set the imaging software to collect images from each selected field of view every 10 minutes for up to 96 hours. Unperturbed mitosis lasts 20 to 40 minutes, and thus 10-minute intervals will provide enough sampling to identify when cells divide. To identify nuclear rupture, which is a transient event, acquire images every five minutes.

Identify cells that enter mitosis by observation of cell rounding, using phase-contrast optics, and/or chromosome condensation, using human histone H2B fused to GFP. Both cell rounding and chromosome condensation are readily visualized by eye. Annotate the time when the cell enters mitosis.

Continue tracking the cell until it reaches its fate. Control cells should efficiently align their chromosomes and enter anaphase within one hour. Visualize anaphase by phase-contrast optics as the cell begins to pinch into two or through visualization of poleward-moving chromosomes labeled with H2B-GFP.

Annotate the time when the cell undergoes anaphase. By contrast, cells treated with paclitaxel will remain rounded with condensed chromosomes for several hours. Identify mitotically arrested cells that undergo cell death.

Cells that die during mitosis are visualized by phase-contrast microscopy, as cells will bleb, shrink, and/or rupture. If imaging H2B-GFP, the chromosomes will also fragment during cell death. Identify mitotically arrested cells that undergo cell slippage.

Cells that undergo mitotic slippage are observed by phase-contrast microscopy, as they flatten back out into interphase and decondense chromosomes without undergoing anaphase. These cells give rise to large tetraploid cells that are often multinucleated. Continue tracking cells from the original field of view.

Once a whole field of view is tracked, move to a separate field of view acquired from the same well and continue tracking the cells. Assess cell fate following mitotic slippage using the non-transformed and chromosomally stable retinal pigmented epithelial cell line expressing the FUCCI system. To confirm that the FUCCI system is working properly, validate that control cells alternate expression of the red and green fluorescent proteins appropriately from analysis of the live-cell imaging data.

Control cells should transition from exhibiting entirely nuclear red fluorescence to exhibiting entirely nuclear green fluorescence during interphase as cells progress from G1 to S phase. Cells should continue exhibiting green fluorescence throughout the completion of mitosis. Immediately following mitosis, cells should once again exhibit entirely red fluorescence during interphase.

Identify cells arrested in mitosis using phase-contrast microscopy, and track them until they undergo mitotic slippage. Cells that slip out of mitosis and back into interphase will change from exhibiting green fluorescence during mitosis to red fluorescence during G1 phase. Track these slipped cells using phase-contrast and epifluorescent imaging by eye to assess their cell fate.

Identify cells that re-enter the cell cycle. These cells are identified by the red-to-green change in fluorescence expression using the FUCCI system that indicates the G1/S transition. Identify cells that undergo G1 cell cycle arrest.

These cells are identified by expression of red fluorescence that persists for greater than 24 hours. Also, identify cells that die in interphase. These cells are identified by cell rupture, blebbing, and shrinking using phase-contrast imaging.

Image the cells as before. The fluorescence signal from the tandem dimer repeat of RFP fused to a nuclear localization signal, or TDRFP-NLS, and H2B-GFP should co-localize during interphase. Mitosis is visualized by cell rounding using phase optics and/or chromosome condensation by H2B-GFP.

Upon mitosis, the nuclear envelope will break down and the TDRFP-NLS signal will become cytoplasmic. Following mitosis, the nuclear envelope will reform in the daughter cells and the TDRFP-NLS signal will become nuclear. Track daughter cells throughout the subsequent interphase by live-cell imaging.

Identify nuclear rupture events by observing a transient burst of nuclear-localized TDRFP-NLS into the surrounding cytoplasm. Within minutes, the nuclear envelope will be repaired and the TDRFP-NLS will be relocalized to the nucleus. Shown here are representative images of a slipped cell turning green and entering S phase after slipping from paclitaxel treatment.

A majority of cells that slipped from paclitaxel treatment underwent a G1/G0 arrest and stayed red for more than 48 hours without undergoing apoptosis. Cells that are red post-slippage will also undergo apoptosis, as represented by this image. Nuclear envelope rupture increases in paclitaxel-treated cells.

TDRFP-NLS and H2B-GFP cells treated with 10-nanomolar paclitaxel have an increased nuclear rupture in which RFP will be delocalized from the nucleus to the cytoplasm in interphase cells. After watching this video, you should have a solid understanding of how to perform long-term live-cell imaging to assess cell fate in response to various drug treatments.