The overall goal of this procedure is to observe anti-cancer therapeutic response in single cells in culture. This method can answer key questions in the cancer research field, including heterogeneous therapeutic response and cell fate. To begin, under a certified sterile laminar flow hood, prepare HT1080 cells in culture-certified dishes according to the text protocol and incubate the cells in a humidified cell culture incubator.
Two days prior to imaging, plate 50, 000 HT1080 FuGENE cells per well into the desired number of wells of a 12-well, no. 1.5 glass bottomed dish. Adjust the number of cells plated depending on their growth rate and the dish used in order to achieve approximately 60%confluence for the start of the experiment.
Set up the environmental chamber to approximately 80%humidity with the temperature at the sample position set at 37 degrees Celsius. Ensure that the water reservoir is filled with sterile distilled water following the manufacturer's directions. Then turn on the environmental chamber to the desired temperature setting, and position it in the microscope stage and set.
If there is any free space between the stage top chamber and stage inset, lay pieces of paraffin film over the edges of the stage inset opening, prior to inserting the chamber into it, to couple the chamber to the stage. Ensure that no connections are pulling on the chamber, both of which can introduce motion artifacts. Insert a dummy dish with water into the chamber, and allow the chamber to equilibrate to 37 degrees Celsius then maintain a stable temperature.
To set up for imaging, turn on the microscope, the computer, and any required peripherals. Depending on the light source, wait to turn it on until needed. With the objective turret in a low position, select the 20x 0.7 numerical aperture objective.
Then position the sample over the objective, which will make it easier to find the cells when the sample is in the chamber. After transferring the cells to be imaged to the environmental chamber, according to the text protocol, seal the chamber to maintain a stable environment and turn on the atmospheric gas. Next, select imaging conditions that will prevent photo toxicity by limiting exposure times and using lower intensity light.
To find the X, Y, and Z planes and the desired wavelengths for each position to be imaged. For long-term time lapse of most cellular processes, acquire one image every ten to twenty minutes. Greater time resolution provides more data points and more robust cell tracking but results in more integrated light exposure and larger data sets.
As HT1080 FuGENE have dynamic green and red phlorescent proteins, use the following exposure times and use a two by two bin. Under advanced settings, enable software-controlled auto-focusing using the default settings. Define an auto-focus range of 10 micrometers with the recommended step size.
In the experimental dish, to reduce thermal drift replace half of the medium with medium containing the desired drug that has been warmed to 37 degrees Celsius. Finally, start the time lapse and acquire images according to the text protocol. These movies show examples of a pair of matched breast cancer-derived MCF7 cell lines that differ only in their p53 status that were treated with an anti-cancer drug that inhibits kinesin-5 resulting in mitotic arrest.
As seen in this figure, when p53 is removed by stable p53 knockdown, instead of arresting after they leave mitosis, the cells go through repeated cycles where they enter a second round of mitosis rather than arresting, and leave mitosis without division. Shown here are cervical carcinoma-derived Hela cells stably expressing the chromatin marker Histone 2b fused to mCherry and beta tubulin fused to EGFP. When treated with the microtubule stabilizing drug paclitaxol, chromosome alignment and segregation is disrupted during mitosis, resulting in the formation of nuclear bulges and micronuclei which are prone to DNA damage.
This cell line stably coexpresses the two phlorescent ubiquitin cell cycle indicators for CDT1, in orange, and geminin in green, and progresses through the cell cycle normally. When the same cells are treated with the CDK46 inhibitor, the cells progress through G2 phase and divide normally then strongly arrest in G1 phase indicating the potential for affective cytostatic effects in growing tumors. The main advantage of this technique is that you can follow a single cell response, directly, in real time over the course of many days rather than infer those responses.
