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A number of estrogen receptor (ER) positive and/or negative breast cancer cell lines are available for research purposes.The validity of the ER status of these cell lines has not been updated.Two estrogen receptor isoforms are known to exist, estrogen receptor alpha (ERα) and estrogen beta (ERβ).Cell lines traditionally known to be ERa negative may be positive for ERβ.This may partly explain the discrepancy in results between different groups of researchers using various experimental procedures.The expression of ER isoforms in breast cancer cell lines was therefore studied to see whether both ER isoforms, as well as any other variants, are expressed at the mRNA and protein levels.Seven breast cancer cell lines: three which are reported as being ER-positive (MCF7, T-47D and ZR-75-1) and four reported as being ER-negative (MDA-MB-231, SK-BR-3, MDA-MB-453 and HCC1954) were investigated.Permeabilised, fixed cells were assessed by flow cytometry (FCM) using two monoclonai anti-ERα antibodies and a polyclonal anti-ERβ antibody.For gene expression studies RT-PCR methodology was applied; primers were used that detect the wild type ERα and ERβ isoforms and several variants.ERα and ERβ proteins were detected by Western blotting.Our results revealed that all the cell lines expressed ERα and ERβ by FCM, with SK-BR-3 showing the least expression.ERa mRNA was not only expressed in ER-positive cell lines but also in ER-negative cell lines including MDA-MB-231 and HCC1954, which also showed weak expression of ER5 and 7 variants.The ERα△3,△5 and △7 spliced variants were present in both the MCF-7 and T-47D ER positive cell lines.