Metabolomics is the study to identify and quantify metabolites present within the cells. To prepare the sample, begin by seeding breast cancer cells along with growth medium in two plates labeled as test and control. Incubate for three days at 37 degrees Celsius. Now, remove the medium and add fresh medium with estradiol in the test plate and plain medium in the control plate.
The estradiol binds to estrogen receptor alpha in breast cancer cells to induce certain expressions, causing a change in the metabolite composition. Incubate the plates for the desired duration. Replace the medium with ice cold phosphate buffered saline or PBS to wash the cells and then add ice cold acetone. The ice cold reagents stop the action of proteases and thus prevent protein degradation.
Now use a scraper to detach the cells from the plates, and transfer them to tubes with the help of a pipette. Take 20 microliters of this cell suspension onto a hemocytometer and count the number of cells. Store the samples at minus 80 degrees Celsius until used for further analysis. In the following protocol, we prepare samples from MCF-7 cells for gas chromatography and mass spectrometry.
Using MCF-7 cells cultured according to the text protocol, add five milliliters of ice cold 1x PBS to the plate. Then tilt the plate several times before removing the PBS. Repeat two more times removing as much PBS as possible after the last wash. Add 750 microliters of prechilled acetone to each plate and scrape the cells, then combine the cells from two plates into a two milliliter tube on ice.
To count the cells for normalization, for each treatment, use two extra plates for cell quantification. Then to detach the cells from the plates add 2 milliliters of HE buffer and incubate for five minutes. Mix the cells thoroughly by pipetting in the HE buffer.
Then use 20 microliters of the cell solution in a hemocytometer to count the cell number under a microscope. Store the samples at negative 80 degrees Celsius before using gas chromatography mass spectrometry analysis to identify and quantify the metabolites. Perform integrative analysis according to the text protocol.