The overall goal of this method is to label and visualize live chromosomes in mitotic cells using Histone 2B-GFP BacMam 2.0 label and a spinning disc confocal microscopy system. This method can be used to visualize mitotic chromosomes in cultured cells to study the effects of genetic modifications or drug treatments on chromosome segregation. The main advantage of this technique is that it provides an easy and convenient method to label and visualize live chromosomes in mitotic cells.
Demonstrating the live imaging procedure will be Doctor Kazuyo Takeda who maintains the Microscopy and Imaging Core Facility in the Division of Viral Products at the FDA Center for Biologics Evaluation and Research. The mouse embryonic fibroblasts, or MEFs, used in this experiment are grown in a two-well chambered cover glass at 37 degrees Celsius and 5%CO2 overnight, as described in the text protocol. On the following day, synchronize the MEFs in G0/G1 phase by incubating in DMEM/F12 growth medium containing 0.1%FBS.
On the day of labeling, prepare a 1.5 milligram per milliliter stock solution of nocodazole in DMSO. A critical step is to calculate the right amount of CL-HB particles per cells to achieve enough labeling for chromosome visualization without toxicity to cells. We have previously determined, by titration experiments, that 30 PPC works best.
Add to the MEFs, 200 microliters of growth medium with 10%FCS, 200 nanograms per milliliter of nocodazole, and CL-HB reagent at 30 particles per cells. Incubate the MEFs at 37 degrees Celsius and 5%CO2 for 18 hours. Visualization of the MEFs is accomplished using a spinning disk confocal microscope system equipped with an environmental chamber and an oil immersion, 63X objective lens.
One day before imaging, turn the environmental chamber power on and warm up the entire chamber at 37 degrees Celsius overnight. On the following day, turn the power on for the microscope stand, camera, spinning disk unit, illuminator, argon laser, computer, and motorized stage. After letting the system warm up for three minutes, start the argon laser by turning on the ignition key.
Switch the toggle switch for the argon laser from standby to laser run. Launch the data acquisition and processing software. Initiate the CO2 controller for the stage top incubator, and set the concentration of CO2 at 5%Remove the chambered cover glass from the incubator, place it on the microscope stage, and select the oil immersion 63X objective lens.
View through the ocular lenses, focus the image, and identify a cell in the stage of nuclear envelope breakdown, or NEBD. Initiate the 488-nanometer argon laser to visualize Histone 2B-GFP. Open the acquisition control window, and set exposure time for the GFP channel.
Identify a cell that is in NEBD. Then, manually determine the cell's top and bottom focal plane, and enter the XYZ optical sectioning settings. Observe the cell for 20 minutes.
It is important to be aware that since not all cells will go through mitosis, mitotic progress must be actively monitored. If the cell does not proceed through cell division after 20 minutes, stop image acquisition and move on to the next cell that is in NEBD. To obtain data for a movie, take pictures every three minutes.
Approximately one hour per cell is needed to image lagging chromosomes during mitosis in PP2A-B56-gamma cells that escaped from the spindle assembly checkpoint. Save images in ZVI file format for further analysis. Live cell imaging was used to visualize the fate of individual cells as they progressed from NEBD through mitosis.
Post-nocodazole treatment, all 21 wild-type cells observed were arrested at metaphase. In contrast, from the 21 cells lacking PP2A-B56-gamma observed, 16 were able to go through mitosis after nocodazole treatment. In addition, abnormal chromosomal segregation was detected in 13 of those MEFs.
An example of a PP2A-B56-gamma cell undergoing chromosomal mis-segregation is shown in this video clip. Once mastered, this technique will take three to four days depending on the cell treatment prior to the labeling protocol. It is important to remember that this is a transient transfection and signals can be detected only up to five days.
This protocol can be used to answer additional questions related to the cell cycle regulation, genomic instability, and the quality of the cell therapy products. After its development, this technique could be used to study live imaging of chromosomes in a wide variety of cell lines, such as primary cell lines, stem cells, neurons, and immortalized T-cells. After watching this video, you should have a good understanding of how to easily label and visualize live chromosomes in mitotic cells using Histone 2B-GFP BacMam 2 label and a spinning disc confocal microscopy system.
