MLE-12 cells were maintained in HITES medium consisting of DMEM/F12 (ATCC, 30-2006), 2% fetal bovine serum (FBS), 0.005 mg/ml insulin, 0.01 mg/ml transferrin (Fitzgerald, 31C-CH1026), 30 nM sodium selenite (Santa Cruz, 253595), 10 nM hydrocortisone (Sigma, H0888), 10 nM β-estradiol, 10 mM HEPES (Gibco, 15630106), 2 mM l -glutamine (Gibco, 25030081), and 1% Pen-Step (Gibco, 15140122). H441 cells were maintained in RPMI-1640 (ATCC, 30-2001) medium supplemented with 10% FBS and 1% Pen-Strep.
β estradiol
Manufactured by Thermo Fisher Scientific
Sourced in United States
β-estradiol is a laboratory reagent that is used for research purposes. It is a naturally occurring steroid hormone that plays a role in the regulation of the estrous and menstrual cycles. β-estradiol can be used in various research applications, such as cell culture studies, receptor binding assays, and hormone analysis.
Automatically generated - may contain errors
Lab products found in correlation
L glutamine, by Thermo Fisher Scientific (2 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions)
Dmem f12, by American Type Culture Collection (1 mentions)
Hepes, by Thermo Fisher Scientific (1 mentions)
Hydrocortisone, by Merck Group (1 mentions)
Pen step, by Thermo Fisher Scientific (1 mentions)
Rpmi 1640, by American Type Culture Collection (1 mentions)
Insulin, by Biosynth (1 mentions)
Pen strep, by American Type Culture Collection (1 mentions)
Dmem f12, by Thermo Fisher Scientific (1 mentions)
Mitomycin c, by Merck Group (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
2 mercaptoethanol, by Thermo Fisher Scientific (1 mentions)
Interleukin 3 (il 3), by Thermo Fisher Scientific (1 mentions)
Lipofectamine ltx, by Thermo Fisher Scientific (1 mentions)
Plus reagent, by Thermo Fisher Scientific (1 mentions)
Bovine insulin, by Thermo Fisher Scientific (1 mentions)
Rpmi 1640 medium without phenol red, by Thermo Fisher Scientific (1 mentions)
4 hydroxytamoxifen, by Merck Group (1 mentions)
9 protocols using β estradiol
1
Maintenance of Lung Cancer Cell Lines
2
Spermatogonial Stem Cell Culture Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
SSCs were cultured on plate with laminin-coated or Sertoli cell feeders. Primary Sertoli cells cultured using DMEM/F12 (Gibco, Grand Island, NY, USA) were supplemented with 10% FBS (Gibco). When Sertoli cells reached about 90% confluency, the cells were treated with mitomycin C (10 mg/l, Sigma, St. Louis, MO, USA) for 3 h and washed five times with PBS. SSCs culture medium consisted of DMEM/F12 (Gibco) supplemented with 1% FBS (Gibco), 30 ng/ml β-estradiol (Gibco), 100 U/ml penicillin, 100 μg/ml streptomycin, 1 × MEM non-essential amino acids, 20 ng/ml GDNF, 10 ng/ml mouse EGF, 10 ng/ml bFGF. The medium was changed every 2–3 days.
The C18-4 cell line was established from type A spermatogonia isolated from 6-day-old mouse testes.43 (link) The cells were maintained in DMEM medium supplemented with 10% fetal calf serum, 1 mM sodium pyruvate, 2 mM glutamine, 50 U/ml penicillin, 50 μg/ml streptomycin and 100 mM non-essential amino acids. The siRNA sequences targeting mouse ESET and Cox4i2 mRNA were designed and synthesized by Genepharma Company (Shanghai, China). The siRNAs were transfected into C18-4 cells using Lipofectamine2000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's protocol.
The C18-4 cell line was established from type A spermatogonia isolated from 6-day-old mouse testes.43 (link) The cells were maintained in DMEM medium supplemented with 10% fetal calf serum, 1 mM sodium pyruvate, 2 mM glutamine, 50 U/ml penicillin, 50 μg/ml streptomycin and 100 mM non-essential amino acids. The siRNA sequences targeting mouse ESET and Cox4i2 mRNA were designed and synthesized by Genepharma Company (Shanghai, China). The siRNAs were transfected into C18-4 cells using Lipofectamine2000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's protocol.
3
Hoxb8-FL Derived Cell Culture
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Parental cells (Hoxb8-FL cells) were cultured in RPMI 1640 medium (Sigma-R8758) supplemented with 10% fetal bovine serum (FBS) (HyCloneTM GE Healthcare), 1% penicillin/streptomycin (Sigma-P0781), 1% l -Glutamine (Sigma-G7513), 0.1% 2-Mercaptoethanol (50 mM stock)(Gibco®), cell culture supernatant from an Flt3L-producing B16 melanoma cell line (5% final concentration) and 1 µM β-estradiol (Sigma-E2758), as originally described16 (link). ME-Parental cell line was cultured as Parental cells. ME-Transformed and MLL-ENL BM were cultured in RPMI 1640 medium (Sigma-R8758) supplemented with 10% FBS (HyCloneTM GE Healthcare), 1% penicillin/streptomycin (Sigma-P0781), 1% l -Glutamine (Sigma-G7513), 0.1% 2-Mercaptoethanol (50 mM stock) (Gibco®) and 10 ng/ml of recombinant murine interleukin 3 (IL-3) (PeproTech). All cell lines were kept at a concentration of 1–10 × 105 cells/ml and the medium was replenished every 1–2 days.
4
Cell Cycle Synchronization Using Estradiol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cell cycle experiments were performed similarly to our previous study16 (link): two sequential 24-h pre-cultures were grown on 1% ethanol minimal medium (EMM) media containing 15 and 10 nM β-estradiol (Alfa Aesar), respectively. To induce cell cycle arrest, cells were filtered, washed and the filter resuspended in estradiol-free EMM (starting OD600 was between 0.1 and 0.2). Before release, inducible LexApr-CLN1 strains were arrested for 15 h. Cells were released into the cell cycle by addition of 200 nM β-estradiol (dissolved at 1 mM in 100% ethanol). Cell cycle arrest and release were verified by bud count at 60 × magnification in a light microscope.
5
Engineered Yeast Strains for Cell Cycle Study
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
All strains were constructed using homologous recombination in a prototrophic Saccharomyces cerevisiae W303 derived background. Plasmid containing NTH1 were constructed using standard molecular cloning. Plasmids were linearized with either SalI in the promoter (full-length constructs) and integrated into the genomic NTH1 promotor or with StuI in the URA3 region (reporter constructs) and integrated into the URA3 locus. All constructs were Sanger-sequenced. Cell cycle inducible strains from our previous study16 (link) were used. Estradiol inducible strains were transformed and maintained on YPD plates containing 100 nM β-estradiol (Alfa Aesar). A detailed strain list is included in the Supplementary Information .
6
QTRT1 Knockout in Breast Cancer Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The highly aggressive breast cancer cell line human MCF7 cells were cultured in a 6-well tissue culture plate in antibiotic-free MCF7 cell growth medium (Eagle’s minimum essential medium with 10% FBS, 10 µg/mL bovine insulin, and 10 nM β-estradiol; Invitrogen, Carlsbad, CA, USA) to 70%–80% confluence. Cells were transfected with 2 µg of QTRT1 Double Nickase Plasmid (sc-413456-NIC), 10 µL LTX Lipofectamine, and 2.5 µL PLUS Reagent (Invitrogen) per well (manufacturer’s protocol). The plasmid encodes a D10A mutated Cas9 nuclease and a unique, target-specific 20-nt guide RNA (gRNA), which has greater specificity of gene knockout than the CRISPR/Cas9 KO plasmid counterpart. QTRT1 Double Nickase Plasmid-derived puromycin resistance gene was used for positive selection of transfected cells. Cells were selected with 1 µg/mL puromycin for 5 days when further cell death was not observed. After selection, cells were collected and serial diluted onto four 96-well plates. Single cells were expanded to obtain individual clones for further study. The culture of MDA-MB-231 cells was as described before [53 (link)], and transfection and selection were as described above.
7
β-estradiol-induced cell proliferation assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
A β-estradiol-induced cell proliferation assay was performed with
steroid-depleted medium, in which RPMI 1640 medium without phenol red
(Invitrogen) was supplemented with 5% charcoal-stripped bovine calf serum
(Thermo Scientific), 1% nonessential amino acids solution, 1 mmol/l sodium
pyruvate, and 0.01 mg/ml bovine insulin. MCF-7 cells were preincubated with
steroid-depleted medium for 6 days, and 2 × 103 of these cells
were re-seeded into 96-well F-bottom plates and incubated for 24 h at 37°C.
β-estradiol (Sigma-Aldrich, final concentration 1 nmol/l) was added followed
by increasing concentrations of KW-2450 or 4-hydroxytamoxifen
(Sigma-Aldrich) and then incubated for 7 days at 37°C. For the
aromatase-dependent cell proliferation assay, MCF-7-Ac1 cells were
preincubated, re-seeded, and treated with steroid-depleted medium according
to the same method as for MCF-7 cells. However, androstenedione
(Sigma-Aldrich, final concentration of 10 nmol/l) was then added followed by
increasing concentrations of KW-2450 or letrozole (Kemprotec, Middlesbrough,
UK) and then incubated for 7 days at 37°C. Cell viability was determined
using Cell Proliferation Reagent WST-1 (Roche Diagnostics, Mannheim,
Germany). The CI and combination effect were determined as previously
described.
steroid-depleted medium, in which RPMI 1640 medium without phenol red
(Invitrogen) was supplemented with 5% charcoal-stripped bovine calf serum
(Thermo Scientific), 1% nonessential amino acids solution, 1 mmol/l sodium
pyruvate, and 0.01 mg/ml bovine insulin. MCF-7 cells were preincubated with
steroid-depleted medium for 6 days, and 2 × 103 of these cells
were re-seeded into 96-well F-bottom plates and incubated for 24 h at 37°C.
β-estradiol (Sigma-Aldrich, final concentration 1 nmol/l) was added followed
by increasing concentrations of KW-2450 or 4-hydroxytamoxifen
(Sigma-Aldrich) and then incubated for 7 days at 37°C. For the
aromatase-dependent cell proliferation assay, MCF-7-Ac1 cells were
preincubated, re-seeded, and treated with steroid-depleted medium according
to the same method as for MCF-7 cells. However, androstenedione
(Sigma-Aldrich, final concentration of 10 nmol/l) was then added followed by
increasing concentrations of KW-2450 or letrozole (Kemprotec, Middlesbrough,
UK) and then incubated for 7 days at 37°C. Cell viability was determined
using Cell Proliferation Reagent WST-1 (Roche Diagnostics, Mannheim,
Germany). The CI and combination effect were determined as previously
described.
8
Endometrial Stromal Cell Co-culture with Blastocysts
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Endometrial stromal cells were isolated and cultured from four biopsies of controls, as described above. ESCs transfected with CDYL shRNA or negative shRNA were grown to confluence in a 24-well plate. Cells were then decidualized by treatment with 10 nM β-estradiol (E2758, Thermo Fisher Scientific), 1 μM progesterone (V900699, Thermo Fisher Scientific), and 1 mM 8-Br-cAMP (ab141448, Abcam) for 48 h. Then hatched blastocysts from C57BL/6 mice with a normal morphology were cocultured with confluent monolayers of decidualized ESCs for 72 h in DMEM/F12 complete medium. The morphological images were captured by microscope TS2 (Nikon, Tokyo, Japan). The trophoblast outgrowth areas were outlined manually and measured using Image J 1.46r.
9
Hyperoxia-induced Lung Cell Damage
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Murine lung epithelial cells (MLE-12) were acquired from ATCC (Manassas, VA, USA) and cultured in Dulbecco's modified eagle medium (DMEM)/F-12 with 2% fetal bovine serum (FBS), 10-nM hydrocortisone, and 10-nM β-estradiol (Thermo Fisher Scientific, Waltham, MA, USA) at 37°C. In order to induce oxygen toxicity, MLE-12 cells were kept in hyperoxic condition with 85% O 2 for 24 h. Cells in the control group were exposed to normoxic condition with 21% O 2 . For cell transfection, MLE-12 cells were seeded in 96-well plates and transfected with plasmid cloning DNA (pcDNA) vector, pcDNA-EDA2R, small hairpin negative control (shNC), shEDA2R (GenePharma, Suzhou, China) via lipofectamine 2000 (Thermo Fisher Scientific) under hyperoxic condition.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!