Live Cell Imaging of Chromosome Segregation During Mitosis

All the experiments conducted in these studies were performed in accordance with protocols approved by the Institutional Animal Care and Use Committee at the Food and Drug Administration (FDA) research facility.

1. Isolation and Culture of Mouse Embryonic Fibroblasts (MEFs)

1. Isolate mouse embryonic fibroblasts (MEFs) from a PP2A-B56γ- mouse strain and wild type littermates by standard protocol ^7,8,9.
2. ExpandMEFs for 3 passages, freeze and store until needed for experiments ⁸.

2. Culturing Mefs in 2-Well Chambered Cover Glass for Live Imaging

1. Prepare 500 mL of MEF growth media containing Dulbecco Modified Eagle Medium (DMEM/F12) with 10% Fetal Bovine Serum (FBS), 1 X Penicillin/Streptomycin antibiotics, L-Glutamine (200 mM) and 1X non-essential amino acids (NEAA) in a 500-mL media bottle.
2. Thaw vials of frozen MEFs from wild type and PP2A-B56γ- mice at passage 3 in pre-warmed water bath at 37 °C.
3. Transfer thawed MEFs to 15-mL tubes and slowly add dropwise 20 mL of DMEM/F12 media to the 15-mL tubes using a 10-mL pipette.
4. Transfer MEFs along with 20 mL of DMEM/F12 growth media in T75 flasks and expand them until the cells are 70% confluent. Confluency is estimated using an inverse microscope at 4x or 10x magnification.
5. Aspirate the growth medium using a glass pasture pipette attached to a vacuum system in the hood, add 3 mL of 0.25% trypsin/EDTA and incubate at 37 °C for 5 min. Add 3 mL of DMEM/F12 growth medium to stop the reaction.
6. Centrifuge the cells at low-speed (300 x g) for 5 min at room temperature. Carefully remove the supernatant and re-suspend the cell pellet in 1 mL of DMEM/F12 growth medium pre-warmed at 37 °C in a water bath.
7. Enumerate cells using the trypan blue exclusion method or other appropriate cell counting method ⁸.
8. Seed approximately 20,000 MEFs/well in a 2-well chambered cover glass. Add 200 µL/well DMEM/F12 growth medium and allow the cells to attach by incubating them overnight at 37 °C and 5% CO₂.

3. Synchronization

1. Synchronize MEFs in G0/G1 phase by incubating cells in DMEM/F12 growth media containing 0.1% FBS for 24 h, to obtain a maximum number of cells in G0/G1 phase.
   Note: Although serum starvation was used as a method of preference, various other methods can be used depending on the mitotic stage at which cells are needed to be arrested ¹⁰.

4. Labeling

1. Prepare 1.5 mg/mL stock solution of nocodazole in DMSO. Nocodazole arrests cells at G2/M phase by inhibiting microtubule formation ¹⁰.
2. Three days post synchronization, add 200 µL of growth medium (with 10% FCS), 200 ng/mL nocodazole and CL-HB regent (Histone 2B-GFP BacMam 2.0).
3. Calculate the CL-HB regent Particles Per Cell (PPC) to be added to cells as follows:
   
   Where the number of cells is the estimated total number of cells at the time of labeling, PPC is the number of Particles Per Cell, and 1 × 10⁸ is the number of particles per mL of the reagent. For example, to label 20,000 cells with a PPC of 30
   
   NOTE: CL-HB regent works well with most cell types between 10 and 50 PPC. However, 30 PPC worked best for this study.
4. Incubate MEFs for 18 h at 37 °C and 5% CO₂.
   NOTE: For the current experiment, visualization of chromosomes in mitotic cells escaping the spindle assembly checkpoint occurred 18 h after cell cycle arrest with nocodazole.

5. Visualization of Lagging Chromosomes

1. Visualize mouse embryonic fibroblasts (MEFs) using a spinning disk confocal microscope system equipped with an environmental chamber and an oil immersion, 63x objective lens.
   1. Use excitation wavelength of 488 nm and emission wavelength 450 nm for GFP channel image acquisition.
   2. Use a spinning disk confocal microscope system with the capability of motorized scanning stages, Z-Piezo inserts, stage-top incubation and direct fluorescence recovery after photo bleaching for this technique.
2. One day before imaging, turn the environmental chamber power ON and warm up the entire chamber at 37 °C overnight.
3. Turn power ON for the microscope stand, camera, spinning disk unit, illuminator, argon laser, computer and motorized stage.
4. Let the system warm up for 3 min; start the argon laser by turning on ignition key. Switch the toggle switch for argon laser from "standby" to "laser run".
5. Launch the data acquisition and processing software.
6. Initiate the CO₂ controller for the stage top incubator and set the concentration of CO₂ at 5%. This must be done before commencement of imaging.
7. Remove the chambered cover glass from the incubator and place on the stage for visualization. Visualize cells via an oil immersion 63x objective lens (NA1.4).
8. View through the ocular lenses, focus the image and identify a cell in nuclear envelope breakdown (NEBD) stage.
9. Initiate appropriate laser (488 nm argon laser to visualize Histone2B-GFP).
10. Open acquisition control window and set exposure time for the GFP channel. Identify a cell that is in NEBD.
    1. Manually determine the target cell's top and bottom focal plane, and enter the xyz optical sectioning settings.
    2. Observe it for 20 min; if the cell does not proceed through cell division, stop image acquisition after 20 min and move on to the next cell that is in NEBD.
11. Approximately 1 h per cell is needed to image lagging chromosomes during mitosis in PP2A-B56γ- cells that escaped from the SAC. To obtain data for a movie, take pictures every 3 min.
    NOTE: Wild type cells arrest and do not progress past NEBD when treated with nocodazole.
12. Save images in zvi file format for further analysis.

6. Image Processing and Analysis

Note: Perform three-dimensional image processing and analysis using any available software such as Axiovision version 4.8.2 or Imaris version 8.2. For this study the ImageJ software was used.

1. Open the image sequence. If the images are already in a stack format, proceed to the next step. If not, combine all relevant images into a stack using 'Image > Stacks > Images to Stack' in the menu bar.
2. Perform any adjustments as required to brightness/contrast and levels.
3. To add a time stamp to the movie:
   1. Go to 'Image > Stacks > Label…' in the menu bar.
   2. Select the appropriate format, the starting time value, and time interval between each image.
   3. Check the 'Preview' box and adjust location and format settings. Press 'OK' to apply the time stamp.
4. To add a scale bar to the movie:
   1. Set the image scaling under 'Analyze > Set Scale' in the menu bar.
   2. In the 'Distance in pixels' field, enter a number of pixels where the distance is known, and in the 'Known distance' field enter the distance. Set the correct unit of length for the distance, e.g., in µm. Press 'OK' to apply the scale to the stack.
   3. Go to 'Analyze > Tools > Scale Bar…' to add the scale bar. Set the size of the bar as 'Width in µm', and adjust the remaining formatting options as appropriate. Check the 'Label all slices' box to add the scale bar to the entire stack.
5. Preview the movie by clicking on the triangular play button on the bottom left edge of the image window. Adjust the frame rate using 'Image > Stacks > Animation > Animation Options' in the menu bar.
6. Export the movie file by selecting 'File > Save As > AVI…' and select the frame rate and compression.
7. Press 'OK' to choose the save location and file name. Press 'Save' to save the movie file.