Live cell imaging enables the observation of dynamic cellular processes over time within a single cell. These approaches are powerful for assessing the conditions that impact the dynamic process of cell division and revealing the impacts that defects in mitosis have on daughter cell proliferation and viability. Phototoxicity that results from light exposure during imaging represents a significant challenge associated with this protocol.
Therefore, exposure times and the imaging interval and duration should be optimized. Using aseptic technique and a biosafety level two safety cabinet, use a sterile disposable glass Pasteur pipette to aspirate the medium from the culture plate containing the cell line carrying the expression construct of interest. Briefly wash the cells with 10 milliliters of sterile PBS with swirling and treat the cells with two milliliters of 0.05%trypsin.
After two to five minutes at 37 degrees Celsius, add eight milliliters of fresh medium to the plate to stop the reaction and resuspend the detached cells with gentle pipetting. Transfer the cell solution to a sterile 15 milliliter tube and collect the cells by centrifugation. Resuspend the pellet in 10 milliliters of PBS and centrifuge the cells again.
Resuspend the pellet in 10 milliliters of medium for counting and dilute the cells to a one to two times 10 to the 5th cells per milliliter of cell culture medium concentration. Then seed 500 microliters of cells into each well of a sterile 12-well imaging bottom plate and place the plate in the cell culture incubator to allow the cells to adhere to the plate's surface. No more than 30 minutes prior to imaging, add a relevant concentration of a mitotic drug to one or more of the wells and add an equal volume of the inhibitor diluent to the same number of wells as the controls.
To prepare the microscope for imaging, place the plate onto the stage of an inverted epifluorescence microscope equipped with a high resolution camera, an environmental chamber preheated to 37 degrees Celsius and a delivery system for humidified 5%carbon dioxide. Select a 20 times air objective with a numerical aperture of 0.5 and equipped for the high contrast fluorescence and phase contrast, or bright field imaging and view the cells to adjust the course and find focus to bring cells into focus. To set the optimal exposure times for bright field, GFP, and RFP image acquisition, select the respective filter cubes with the appropriate excitation and emission for the fluorophores that will be imaged and click Play.
If the signal is not sufficiently intense, adjust the auto exposure and in each fluorophore channel select the appropriate binning from the Format dropdown menu. Select and calibrate the microscope stage to the multi-well dish according to the manufacturer's instructions and use the image acquisition software to highlight or otherwise select the wells that will be imaged. Open the generated points control panel and under Working Area select Restricted and Border to restrict the coordinate selection area to exclude the boundaries of the well.
In the Area Restriction dropdown menu, select Whole Area and select a random point placement. Set the count to six and click Randomize to select the number and distribution of the points to be captured per well, respectively. Then click on and enter the values in the time sequence control panel to select and input the time interval and the duration for collecting the images.
To visualize the RFP-H2B labeled chromatin, select the images captured with the RFP filter cube in place and identify a cell entering mitosis as indicated by the initial chromatin compaction and nuclear envelope breakdown to determine the mitotic timing of the metaphase alignment and anaphase onset in individual cells. Track the cell of interest through consecutive time points in the acquired movie to determine the number of time points or minutes from mitotic entry until RFP-H2B labeled chromatin completes the alignment at the cell equator during metaphase. To monitor the mitotic timing, mitotic fidelity, and cell fate, continue to track the cell through consecutive time points to identify the time coordinate at which anaphase chromosome segregation is apparent and/or when chromatin decompaction and nuclear envelope reformation has occurred.
Then visualize RFP-H2B to identify cells in each population that exhibit mitotic defects, including lagging chromosomes and chromatin bridges during anaphase chromosome segregation. By visualizing alpha tubulin-EGFP, it can be observed that cells that experience spindle disruption undergo dynamic changes as spindle pull focusing is achieved and a bipolar mitotic spindle is formed in preparation for cell division. Concurrent with spindle assembly, chromosome movement can be visualized with RFP-H2B to assess the chromosome alignment and segregation fidelity.
Using live cell imaging approaches, these representative results show that cells with a normal centrosome content are able to proceed from nuclear envelope breakdown through metaphase alignment and anaphase onset to achieve a bipolar division in under 30 minutes. In the presence of extra centrosomes, nearly 50%of the cells are able to overcome a transient multipolar mitotic spindle and to form a bipolar spindle in complete cell division. The remaining cells are unable to achieve a bipolar spindle and as a result, exit mitosis through a multipolar division.
Regardless of whether spindle bipolarity is achieved, cells with extra centrosomes exhibit a significantly increased duration of mitosis compared to cells with two centrosomes. Indicating that the dynamics of mitotic progression may be altered even when changes in mitotic outcome are not apparent. When defining exposure times for fluorescent channels, optimize the exposure times to limit phototoxicity.
Also camera pixel binning can be selected to allow lower exposure times to be used. This procedure has applications in pharmacological screenings, invasion, and migration analysis. These approaches can also be employed to examine the efficacy of drug treatments or the dynamics of cellular motility.
