The overall goal of this experiment is to measure microtubule dynamic instability in docetaxel resistant breast cancer cells. This method can help answer key questions in the field of cell biology about microtubule dynamic instability under physiological conditions, and following various drug treatments. The main advantages of this technique are that it provides a simple, reliable and physiologically applicable protocol for studying microtubule dynamics in living cells.
Before beginning the procedure, grow the breast cancer cells in complete culture medium in 10 centimeter culture dishes at 37 degrees Celsius, and 5%carbon dioxide to the appropriate degree of confluency. The day before the experiment, rinse 24 millimeter poly L-lysine coated glass cover slips with 70%alcohol and expose the slips to UV light overnight. The next morning, place one cover slip into each well of a six well plate.
Then wash the cultures with PBS, and detach the cells in each dish with 0.5 milliliters of trypsin EDTA. When the cells have begun to lift from the plate bottom, neutralize the trypsin with five milliliters of fresh culture medium and count the cells. Then add approximately one times 10 the fifth cells to each cover slip, shake the plate gently, and return the cells to the incubator for at least 36 hours.
To assay the microtubule dynamics of the cells mix GFP tagged alpha tubulin stock solution several times by inversion to ensure a homogenous solution without vortexing. Then replace the culture medium in each well with two milliliters of fresh medium containing 40 microliters of the tag tubulin. Shake the plate gently to allow complete mixing of the marker with the culture medium and return the plate to the incubator for another 24 hours.
The next day, replace the culture medium with fresh medium containing docetaxel and return the plate to the incubator for 30 minutes. While the cells are being inhibited, turn on the inverted fluorescence microscope, and prewarm the sample holder to 37 degrees Celsius. Then select the 60X 1.42 NA oil objective lens and mount the first cover slip onto the sample holder.
Cover the sample with one milliliter of fresh 37 degree Celsius docetaxel containing medium and locate a cluster of flat cells exhibiting a high GFP tagged alpha tubulin fluorescence intensity with clearly visible microtubule structures. Focus on the peripheral area of one of the cells in the cluster, and set the appropriate exposure time in the image acquisition frequency. Exactly 50 minutes after adding the first aliquot of docetaxel begin recording the microtubule dynamics for two minutes, obtaining a photo every two seconds.
When all of the images have been acquired, image the next cell as just demonstrated, until images for five cells total from all three wells of each docetaxel concentration have been captured. When all of the images have been acquired for each inhibitor concentration open the deconvolution program, within the microscope system and click process. Next, select deconvolve and drag the image file to the input window.
Click do to deconvolve the image, and open the deconvolved image file within the microscope software. Click file and save as movie. Set the movie format as AV and the animation style as forward.
Set the compression quality to 100%and the frame rate to five per second. Then check the animate through time box and click do it to generate the video. In this representative experiment, normal and docetaxel resistant breast cancer cells were treated with docetaxel and imaged as just demonstrated, but the effects of the treatment on microtubule growth and shortening, most evident in the non docetaxel resistant cells.
Indeed, measuring the rate of microtubule growth and shortening under 0.5 micromolar docetaxel treatment conditions reveals a strong inhibition of the tubulin dynamics of parental but not docetaxel resistant cells. Once mastered, the entire process can be completed within 60 hours if the techniques are performed properly. While attempting this procedure, it is important to remember to select flat cells, with a high GFP tubulin fluorescence intensity, and to reduce both the intensity of the excitation fluorescence and the duration of exposure to avoid fading of the GFP fluorescence.
Since it's development, this technique has paved the way for researchers in the fields of cell and cancer biology to assess the effects of various microtubule targeting agents on normal and cancerous cells. After watching this video, you should have a good understanding of how to measure microtubule dynamic instability in living cells under a variety of conditions.
