An amount of 5 ml of MSM medium containing phenol which showed bacterial growth was transferred to a sterile tube and the pH was adjusted to 8.0, then subjected to centrifugation at 2700g for 20 min.
In these studies, however, cell cultures were grown in a medium containing phenol red and foetal bovine serum, two sources of exogenous estrogenic compounds [ 20- 23].
Standard BG1Luc cell culture maintenance medium is complete growth medium containing phenol red, FBS and various additives and thus contains estrogen and chemicals with EA that facilitate cell growth.
However, in this study, the cell cultures were grown in a medium containing phenol red and foetal bovine serum, two sources of exogenous oestrogenic compounds (Butler et al, 1981; Page et al, 1983; Berthois et al, 1986; Germain and Harbrioux, 1993).
The human breast carcinoma cell line MCF-7 (ER+) was obtained from the American Type Culture Collection LGC Promochemm, Teddington, UK) and maintained in Dulbecco's minimal essential medium containing phenol red, supplemented with 10% fetal calf serum and antibiotics (Sigma, Poole, Dorset, UK).
MDA-MB-231 (breast ER−ve), PC-3 (prostate AR−ve), MCF-7 (breast ER+ve), A2780 (ovarian ER−ve) and LNCaP (prostate AR+ve) cancer cells were obtained from the American Type Culture Collection LGC Promochemm, Teddington, UK) and maintained in Dulbecco's minimal essential medium containing phenol red, supplemented with 10% fetal calf serum and antibiotics (Sigma, Poole, UK).
On the following day, the medium was shifted to estrogen-free medium containing phenol-red-free RPMI (Invitrogen) and 5% charcoal-dextran-stripped human serum (Hyclone Laboratories) with controls or test material.
Before treatment, cells were incubated for 24 h in serum free medium containing phenol-free DMEM supplemented with 1 mM sodium pyruvate, 4 mM L-glutamine, 1 100 nonessential amino acids, 1% insulin-transferrin-selenium-X supplement, 0.1% BSA, and antibiotics as described above.
The culture medium contained phenol red-free D-MEM (Nacalai Tesque, Kyoto, Japan), 1× Glutamax (Life Technologies, Carlsbad, CA), 1 × B-27 supplement (Life Technologies), 10 mM HEPES, 200 μM beetle luciferin (Promega KK, Tokyo, Japan), 100 units/mL penicillin and 100 μg/mL streptomycin (Nacalai Tesque).
Culture medium consisted of minimum essential medium (MEM) containing phenol red (Sigma, A-6770) and supplemented with 10% fetal bovine serum (FBS), penicillin (10 U ml−1), streptomycin (10 μg ml−1), amphotericin B (0.25 μg ml−1), L-glutamine (2 m M) and sodium pyruvate (1 m M).
Medium and buffers containing phenol red were avoided when collecting imaging data.
