Background & objective: Quantitative and qualitative detection of estrogen receptor (ER) and progesterone receptor (PR) levels are very important for predicting prognosis and evaluating the outcome of endocrine therapy of breast cancer patients. Western blot analysis and Flow cytometry (FCM) are important means for quantitative and qualitative analysis of proteins, but conventional Western blot analysis need to extract proteins from fresh cells. The present study was designed to establish Western blot analysis of human estrogen receptor (ER) and progesterone receptor (PR) in fixed breast cancer cells, and to explore the possibility of ER and PR analysis in fixed cells by flow cytometry and Western blot analysis simultaneously.
Methods: Proteins extracted from fresh and fixed cells of different exponentially growing breast cancer cell lines were labelled using 1D5 and PgR636, which were monoantibodies to ERalpha and PR, respectively. The expression of ER and PR were determined using Western blot analysis, and the results were compared with that of fixed cells measured with flow cytometry.
Results: Clear and correct ERalpha bands were observed with Western blot analysis in cell lines T-47d, MCF-7, and ZR-75-1, and the band density of fixed T-47d and ZR-75-1 was higher than that of fresh cells. The expression of ERalpha in MM231 cells was negative. Clear and correct PR bands were visible in T-47d and ZR-75-1 cells with Western blot analysis, and the band density of fixed cells was higher than that of fresh cells. And the expression of PR in MM231 and MCF-7 cells were negative. In addition, positive expression of ER and PR in different cell lines measured by flow cytometry were the same with that analyzed by Western blot analysis.
Conclusion: After fixed with 0.25% paraformaldehyde and 75% ethanol, breast cancer cells can be used for not only quantitative measurement of ER and PR, but also Western blot analysis of ER and PR.