Ectopic ESCs were cultured in 24-well plates (Corning, Steuben County, NY, USA) at a density of 1 × 105 cells/well. The co-culture systems were established by incubating 2 × 105 monocytes with ESCs or alone, adding of estrogen (10−8 M; Sigma), 1-MT (0.05 mM; Sigma), or estrogen (10−8 M; Sigma) plus 1-MT (0.05 mM; Sigma). Meanwhile, naive CD4+ T cells were cultured in 24-well plates that coated with monoclonal anti-CD3 (5 μg/ml; Biolegend) and monoclonal anti-CD28 (1 μg/ml; Biolegend), in the presence of recombinant human IL-2 (50 ng/ml; Biolegend). The monocytes-derived macrophages and 2 × 105 naive CD4+ T cells were collected to co-culture with ESCs in 1 ml medium/well for 5 days (Supplementary Figure 1 ).
Estrogen
Manufactured by Merck Group
Sourced in United States, China, Germany
Estrogen is a lab equipment product used for the quantitative analysis of estrogen levels in biological samples. It is a sensitive and specific tool for researchers and clinicians to measure estrogen concentrations, which is crucial for various applications in the fields of reproductive biology, endocrinology, and clinical diagnostics.
Automatically generated - may contain errors
Lab products found in correlation
Progesterone, by Merck Group (8 mentions)
24 well plate, by Corning (3 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (3 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (3 mentions)
Mda mb 231, by Thermo Fisher Scientific (2 mentions)
Mda mb 231, by American Type Culture Collection (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Mcf 7, by American Type Culture Collection (2 mentions)
Mcf 7, by Thermo Fisher Scientific (2 mentions)
Ethanol, by Merck Group (2 mentions)
Ryanodine, by Bio-Techne (2 mentions)
Recombinant human il 2, by BioLegend (1 mentions)
Monoclonal anti cd3, by BioLegend (1 mentions)
Monoclonal anti cd28, by BioLegend (1 mentions)
Gemcitabine, by Merck Group (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Abi prism 7900 sequence detector, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Harvard Bioscience (1 mentions)
Pregnyl, by Organon (1 mentions)
Bt474, by American Type Culture Collection (1 mentions)
37 protocols using estrogen
1
Ectopic ESCs Co-culture Assay
2
Investigating the Effects of Estrogen and Combination Therapies on Breast Cancer Metastasis in Mice
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Female Balb/c mice (4 weeks of age) were obtained from the Experimental Animal Center of Wuhan University (Wuhan, China) and housed in the Experimental Animal Center of Tongji Medical College (Wuhan, China). All female Balb/c mice underwent bilateral oophorectomy under chlorpromazine anesthesia. After a week-long recovery, the Balb/c mice were injected subcutaneously with one million 4T1 cells and EMT6 cells resuspended in PBS. During the next two weeks, mice in the different groups were given a subcutaneous injection of (1) PBS (blank control group), (2) estrogen (100 μg/kg, Sigma Aldrich, USA), (3) estrogen and AMD3100 (5 mg/kg, Sigma Aldrich, USA), or (4) estrogen and gemcitabine (Sigma Aldrich, USA). Mice in the first three groups were injected every day. Mice in the final group were given a intraperitoneal injection of a single dose of 100 mg/kg gemcitabine one day before the cells were transplanted, and estrogen was injected every day thereafter as previously described. The tumor tissues were obtained from tumor-bearing mice after they were sacrificed.
3
Quantifying mRNA Expression in ESCs
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Total RNA from ESCs treated with estrogen (10−8 M; Sigma), 1-MT (0.05 mM; Sigma), estrogen (10−8 M; Sigma) along with 1-MT (0.05 mM; Sigma), or knockdown of MRC2 with shRNA for 48 h was extracted using the Trizol reagent (Life Technologies, Carlsbad, CA, USA), according to the manufacturer instructions. Total RNA (2 μg) was reverse transcribed into first-stand cDNA (TaKaRa Bio Inc., Japan) following the manufacturer protocol, which was then used as a template for polymerase chain reaction (PCR) amplification. Real-time PCR was performed using ABI PRISMTM 7900 Sequence Detector (Applied Biosystems, Warrington, UK). The primers sequences used are listed in Table 1 . PCRs was carried out for 40 cycles using the following conditions: denaturizing at 95 °C for 30 s, annealing at 95 °C for 5 s, and elongation at 60 °C for 34 s. The expression levels of the samples were expressed as arbitrary units defined by the 2−ΔΔCT method. All measurements were performed in triplicate. The specificity of the product was assessed by melting curve analysis.
4
PBMC Stimulation to Analyze Y-P30/Dermcidin
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5 × 106 PBMCs from non-pregnant women were first cultured for 24 h in RPMI 1640 medium (Invitrogen, Karlsruhe, Germany) supplemented with 100 ng/ml penicillin/streptomycin and 3% of charcolized fetal bovine serum (FBS) (Biochrom, Berlin, Germany) to reduce undesirable side effects due to hormonal contaminations of FBS. Afterwards, the cells were stimulated for 1 h with 50 μg/ml alpha-feto-protein (Antikoerper-Online, Aachen, Germany), 100 IU/ml human chorionic gonadotropin (Pregnyl, Organon, Netherlands), 10 ng/ml progesterone (Sigma, Steinheim, Germany), 100 pg/ml estrogen (Sigma, Steinheim, Germany), or progesterone and estrogen in combination. PBMCs cultured alone served as controls. After stimulation PBMCs were harvested, washed twice in PBS and frozen as cell pellets for Y-P30/dermcidin expression analysis by PCR.
5
Breast Cancer Cell Lines Cultivation
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Human breast cancer cell lines MCF-7, T-47D, BT-474, and MDA-MB-231 cells were obtained from ATCC (Manassas, VA, USA). MCF-7 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (FBS; Gibco). T-47D, BT-474, and MDA-MB-231 cells were cultured in RPMI-1640 medium (Gibco) supplemented with 10% FBS. Cells were cultured at 37 °C with 5% CO2. The immortalized non-tumorigenic human breast epithelial cell line MCF10A was purchased from ATCC and cultured in DMEM/F12 medium supplemented with 5% horse serum, 20 ng/mL epidermal growth factor (EGF), 10 µg/mL insulin, 0.5 µg/mL hydrocortisone, and 100 ng/mL cholera toxin.
In experiments involving treatment of cells with estrogen, which was purchased from Sigma-Aldrich (St. Louis, MO, USA), ER+ breast cancer cells were cultured in phenol red-free medium supplemented with 5% charcoal dextran-stripped FBS for 24 h, and estrogen was added.
In experiments involving treatment of cells with estrogen, which was purchased from Sigma-Aldrich (St. Louis, MO, USA), ER+ breast cancer cells were cultured in phenol red-free medium supplemented with 5% charcoal dextran-stripped FBS for 24 h, and estrogen was added.
6
Simvastatin and Estrogen Preparation
7
Breast Cancer Cell Line Culturing and Estrogen Treatment
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MCF7, T47D and MDA-MB231 cell lines purchased from NCCS (Pune, India) were maintained as previously described.66 (link) MCF10A, a kind gift from Dr Annapoorni Rangarajan (IISc, Bangalore, India) was maintained as previously described.67 (link) The cells maintained for 48–72 h in phenol-free medium prior to hormone treatments were then treated with estrogen (Sigma, St Louis, MO, USA) at a concentration of 100 nM for 24–48 h. During inhibition studies, MCF7 cells were treated with 10 μM of tamoxifen and 1 μM of ICI182,780 (Sigma) for 6 h prior to hormone treatment. To study the direct effect of estradiol in CASP7 expression, MCF7 cells were treated with 10 μg/ml of cyclohexamide and 5 μM of actinomycin D (Sigma) for 30 min followed by hormone treatment for 24–48 h.
8
Chondrocyte Estrogen Receptor Modulation
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According to the methods described in Ref. 13, cells or explants were serum-starved overnight prior to stimulation. Chondrocyte cultures were supplemented with physiological concentrations estrogen (synthetic; 0.1nM; Sigma-Aldrich, shanghai, China), and with or without estrogen receptor inhibitors-ERαinhibitor MPP25 (link) (20uM; Sigma-Aldrich, shanghai, China) and ERβ inhibitor PHTPP26 (link),27 (20uM; Sigma-Aldrich, shanghai, China). Later, recombinant human TGF-β1 (10 ng/mL; Peprotech, shanghai, China) and IL-1β (1 ng/mL; Peprotech, shanghai, China) were added into the culture medium, respectively. Then, E2 (1 nM) was added into the training systems including TGF-β1 or IL-1β. DMSO (Sigma-Aldrich, shanghai, China) was used as vehicle control in the experiments. And chondrocytes were pre-incubated with inhibitors for 0.5 h prior to the addition of E2. estrogen was added 30 min prior to TGF-β1 and IL-1β. Cultures were maintained for 24 h, at which point medium was harvested for ELISA, chondrocytes were harvested for qPCR or WB.
9
Neonatal Estrogen Exposure and Ovariectomy
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C3H strain mice purchased from Clea Japan (Tokyo, Japan) were kept at the Laboratory Animal Resource Center of the University of
Tsukuba. They were housed in a plastic cage in standard laboratory animal facilities with controlled lighting (14 h/day), the
temperature controlled to 25 ± 1 C and food and water provided ad libitum. All experimental protocols involving
animals conformed to the Tsukuba University Guidelines for Care and Use of Laboratory Animals.
Neonatal administration of estrogen and ovariectomy in adulthood were performed basically as previously described [12 (link)] with minor modifications. Briefly, neonatal mice were subcutaneously administrated a daily
single shot of E2 (20 µg), DES (1 µg) or the vehicle (20 µl of sesame oil per head) alone for 4 days starting from day
1, the first day when the pup was identified (at 1000 h). In some cases 1,25(OH)2D was added to the DES solution or the
vehicle from concentrated stocks dissolved in ethanol to give the final amount described in the Results. The mice were
ovariectomized at day 35, and the vagina was excised at day 42 of age.
All chemicals including vitamin D and estrogen were purchased from Sigma-Aldrich Japan (Tokyo, Japan) unless otherwise noted.
Tsukuba. They were housed in a plastic cage in standard laboratory animal facilities with controlled lighting (14 h/day), the
temperature controlled to 25 ± 1 C and food and water provided ad libitum. All experimental protocols involving
animals conformed to the Tsukuba University Guidelines for Care and Use of Laboratory Animals.
Neonatal administration of estrogen and ovariectomy in adulthood were performed basically as previously described [12 (link)] with minor modifications. Briefly, neonatal mice were subcutaneously administrated a daily
single shot of E2 (20 µg), DES (1 µg) or the vehicle (20 µl of sesame oil per head) alone for 4 days starting from day
1, the first day when the pup was identified (at 1000 h). In some cases 1,25(OH)2D was added to the DES solution or the
vehicle from concentrated stocks dissolved in ethanol to give the final amount described in the Results. The mice were
ovariectomized at day 35, and the vagina was excised at day 42 of age.
All chemicals including vitamin D and estrogen were purchased from Sigma-Aldrich Japan (Tokyo, Japan) unless otherwise noted.
10
Validating ER-alpha Target Genes
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RT-PCR was performed follow the procedure mentioned before, the primers of these eight randomly selected overlapped genes were listed in Table S8 . The validation was carried in two breast cancer cell lines, including ERα transgenic and wide type MDA-MB-231 cells, and 100 nM Estrogen (Sigma, St. Louis)-stimulated and wide type MCF-7 cells. For further verifying the relationship between ERα and target genes at clinical level, GEPIA (http://gepia.cancer-pku.cn/index.html ), which is an interactive web server for analyzing the RNA sequencing expression data of 9736 tumors and 8587 normal samples from the TCGA and the GTEx projects, was proceed using a standard processing pipeline [63 (link)]. Pearson correlation analyses between ERα and target genes were performed based on the TCGA Breast invasive carcinoma (BRCA) database. And we used the non-log scale for calculation and the log-scale axis for visualization. A p value < 0.05 was considered to be statistically significant.
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