This video describes a technique of preparing breast cancer cell samples for metabolite profiling. Breast cancer cells are treated with estradiol, which binds to the estrogen receptor alpha and causes change in the metabolite composition.
1. Metabolomics Assay Sample Preparation
- Cell Culture and Treatment
- Seed 400,000 MCF7 cells per 10 cm plate in growth medium. For each treatment prepare three samples. Use two plates in each sample to receive enough cells for the detection. On day 3, remove the medium and add 5 ml fresh medium with or without 10-8 M Estradiol or E2 to the cells. Treat the cells for 24 hr (37 °C in humidified 5% CO2 atmosphere).
- Sample Collection
- Add 5 ml ice cold 1x PBS to the plate, aspirate the PBS after briefly tilting the plate several times. Repeat twice, and remove as much PBS as possible after the last wash.
- Add 750 µl of pre-cold acetone to each plate and scrape the cells. Combine the cells in acetone from two plates into a 2 ml tube on ice.
- Cell count for normalization
- For each treatment, use two extra plates for cell quantification. To de-attach the cells from plate, add 2 ml of HE buffer (1x HBSS, 10 mM HEPES, 0.075% NaHCO3, and 1 mM EDTA) into each plate and incubate for 5 min.
- Collect and mix well the cells by pipetting cells in the HE buffer. Use total of 20 µl cell solution to count the cell number using a hemocytometer.
- Store the samples at -80 °C before sending to the Metabolomics center to identify and quantify the metabolites using Gas chromatography mass spectrometry (GC-MS) analysis.