The E-screen assay: a comparison of different MCF7 cell stocks.

Abstract

For routine maintenance, cells were grown in Dulbecco's modification of Eagle's medium (DME) supplemented with 5% fetal bovine serum (FBS; PAA Labor und Forschungs Ges, MBH, Linz, Austria) in an atmosphere of 5% C02/95% air under saturating humidity at 37°C, except for MCF7 cells BB104, which were routinely maintained in 10% charcoal dextran-treated human serum (CDHS)-supplemented phenol red-free DME medium, prepared as described below.Plasma-derived human serum was prepared from expired plasma by adding calcium chloride to a final concentration of 30 mM to facilitate clot formation. Sex steroids were removed from serum by charcoal-dextran stripping (6).Cellproliferation experiments. We used MCF7 cells in the E-screen test according to a technique slightly modified from that originally described by Soto et al. (5). Briefly, cells were trypsinized and plated in 24-well plates (Limbro, McLean, Virginia) at an initial concentration of 10,000 cells per well in 5% FBS in DME. BB104 cells were seeded in 10% CDHS supplemented medium. The cells were allowed to attach for 24 hr, then 10% CDHS-supplemented phenol red-free DME was substituted for the seeding medium. A range of concentrations of the test compound were added, and the assay was stopped after 144 hr by removing the medium from wells, fixing the cells, and staining them with sulforhodamine-B (SRB).The fixation and staining technique was modified from that described by Skehan et al. (9). Briefly, cells were treated with cold 10% trichloracetic acid and incubated at 4°C for 30 min. Then the cells were washed five times with tap water and left to dry. The fixed cells were stained for 10 min with

Candida albicans morphogenesis is influenced by estrogen

“…We were able to remove a factor or factors from the serum by treatment with activated charcoal, although it was not within the scope of the present study to attempt a biochemical analysis of the compounds removed from the serum. Charcoal treatment of serum is not a specific procedure and may remove components other than estrogen, but its effectiveness in removing estrogen is clearly revealed by the loss of estrogenic activity in charcoal-treated serum as indicated by highly sensitive bioassays including the E-screen which uses cell cycling of human MCF-7 breast cancer cells [14] or the YES (yeast estrogen screen) assay in which the human estrogen receptor and a reporter consisting of two copies of estrogen response elements fused to the lacZ gene are cloned into Saccharomyces, making it responsive to natural and artificial estrogens [15,16]. Our findings revealed that at least part of the germ tube stimulating activity was restored by the addition of beta estradiol to charcoal-stripped serum.…”

Salmon Eggshell Protein Expression: A Marker for Environmental Estrogens

“…In this communication we report the complementary DNA cloning of two eggshell protein variants from Atlantic salmon and show that specific transcripts are present in large amounts in the liver of juvenile fish treated with E 2 or the environmental estrogen mimetic, 4-nonylphenol (NP) (Nimrod and Benson, 1996). Present in different industrial surfactants as a degradation product of alkylphenol ethoxylate, NP is documented to possess estrogenic function in the E-screen assay (Villalobos et al, 1995;Ren et al, 1997), in recombinant yeast assay (Gaido et al, 1997), and in trout hepatocytes (Flouriot et al, 1995;Lech et al, 1996).…”

Synthesis and Anticancer Activity of the (R,S)‐Benzofused 1,5‐Oxathiepine Moiety Tethered to Purines through Alkylidenoxy Linkers

“…The product obtained was then purified by preparative chromatography (MeOH/Cl 2 CH 2 : 0.3:10) to give 22 as a pale-yellow oil (96 mg, 57 %). Microwave-assisted synthesis of [13][14][15][16][17][18][19][20]. DIAD (1.1 mmol) was added dropwise to a suspension of 21 or 22 (1 mmol), triphenylphosphine (1.1 mmol), and 6-chloropurine (1 mmol) in dry THF (10 mL mmol À1 ), cooled in an ice bath, under argon.…”

“…[20] Aliquots of MCF-7 cell suspension (1 10 3 cells per well) were seeded into 24-well plates and incubated for 24 h. The cells were then treated with various concentrations of drugs in the culture medium. Three days later, the wells were aspirated, fresh medium and treatment were added, and cells were maintained for three additional days.…”

“…Three days later, the wells were aspirated, fresh medium and treatment were added, and cells were maintained for three additional days. Thereafter, cells were processed as described previously, [20] using a Titertek Multiscan apparatus (Flow, Irvine, CA, USA) at 492 nm. We evaluated the linearity of the SRB assay with a cell number for each cell line before each cell growth experiment.…”