The glucose metabolism-associated inhibitor STF-31 (S7931), AKT signaling inhibitor MK2206 (S1078), and IGF1R tyrosine inhibitor AG-1024 (S1234) were purchased from Selleckchem (Houston, TX); and ROCK inhibitor (ALX-270-333-M025) was got from Enzo Life Sciences (USA). LY294002 (440204) and Oligomycin (495455) were purchased from Calbiochem (Germany). 2-DG (D8375) was purchased from Sigma (St. Louis, Missouri, USA). Fluorescein isothiocyanate-gelatin was obtained from Life Technologies (G13187).
Stf 31
Manufactured by Selleck Chemicals
Sourced in United States
The STF-31 is a laboratory equipment designed for the filtration and separation of chemical solutions. It utilizes a stainless steel filtration system to efficiently remove particulates and impurities from liquid samples.
Automatically generated - may contain errors
Lab products found in correlation
Ag 1024, by Selleck Chemicals (1 mentions)
Mk 2206, by Selleck Chemicals (1 mentions)
Catalase, by Merck Group (1 mentions)
Mmessage mmachine sp6 kit, by Thermo Fisher Scientific (1 mentions)
Napabucasin, by Selleck Chemicals (1 mentions)
Interleukin 6 (il 6), by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Rapamycin, by Selleck Chemicals (1 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
α mem, by Thermo Fisher Scientific (1 mentions)
Marizomib, by Cayman Chemical (1 mentions)
Mcf 10a, by American Type Culture Collection (1 mentions)
5 protocols using stf 31
1
Metabolic and Signaling Pathway Inhibitors
2
Zebrafish Developmental Manipulation Protocols
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All MOs were ordered from Gene Tools (USA). MOs were injected at one-stage embryos to generate morphants at a dose of 2.44 ng for cebpγ, 2 ng for nkx2.5, and 1.83 ng for tal1. For mRNA synthesis, full-length complementary DNA (CDS) was cloned into pCS2+ hemagglutinin (HA) vector. Capped mRNA was synthesized using the mMESSAGE mMACHINE SP6 Kit (Ambion). The mRNAs were injected into one–cell stage zebrafish embryos at 150 pg per embryo for mRNAcebpγ, 400 pg per embryo for mRNAHA-atf3, and 200 pg per embryo for mRNAtal1. Catalase (C3556, Sigma, USA) was injected at 350 ng per embryo. MO sequences and the primers used for mRNA synthesis are listed in table S2. d -Glucose was applied to the zebrafish embryos from fertilization to 9 ss, 24 or 72 hpf at a dose of 1 or 2%. STF-31 (S7931, Selleckchem, USA) was applied to the zebrafish embryos from fertilization to 9 ss at a dose of 10 μM.
3
Evaluating Anti-Cancer Compound Efficacy
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Napabucasin, STF‐31, and 2‐DG were purchased from Selleck (Selleckchem, Houston, TX, USA). Napabucasin and STF‐31 were dissolved in DMSO at 50 mm as a working stock solution, and 2‐DG was dissolved in saline. IL‐6 was purchased from PeproTech (Cranbury, NJ, USA) and stored at –80 °C. The functional grade CD47 monoclonal antibody (B6H12) was purchased from eBioscience (San Diego, CA, USA) and stored at 4 °C.
4
Macrophage and Pre-osteoblast Cell Culture
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The RAW 264.7 macrophages and MC3T3-E1 pre-osteoblasts (The Shanghai Cell Bank of the Chinese Academy of Sciences, China) were used in this study. RAW 264.7 cells were cultured in DMEM (Gibco, USA) that contains 10% fetal bovine serum (Gibco, USA) and 1% penicillin/streptomycin in a humidified atmosphere containing 5% CO2 at 37°C. The culture medium was changed every day. The cells were passaged by dislodging from the flask surface using a cell scraper. An inhibitor of autophagy (3-MA; 1 mM, Selleck, USA), activator of autophagy (rapamycin; 100 nM, Selleck, USA), the inhibitor of GLUT1 (STF31; 10uM, Selleck, USA) and PI3K activator (740Y-P; 10 uM, Selleck, USA) were used. MC3T3-E1 pre-osteoblasts were maintained in α-MEM (Gibco, USA) containing 10% fetal bovine serum and 1% penicillin–streptomycin in a humidified atmosphere containing 5% CO2 at 37°C. The culture medium were changed every 3 days, and the cells were passaged upon reaching 80% confluence by dislodging from the flask surface using trypsin.
5
Breast Cancer Cell Line Authentication and Treatment
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All breast cancer cell lines and non-malignant mammary epithelial cell line (MCF10A) were obtained from the American Type Culture Collection (ATCC). The 4T1.2 and 4T1BR4 cell lines were kindly provided by Dr Norman Pouliot, Olivia Newton-John Cancer Research Institute, Australia. All breast cancer cell lines were cultured in DMEM media supplemented with 10% fetal bovine serum (FBS). D492 mammary epithelial cells were provided by Dr Thorarinn Gudjonsson (The Panum Institute, Denmark) and were maintained as described previously 15 (link). All cell lines were tested for Mycoplasma infection and authenticated using short tandem repeat (STR) profiling by scientific services at QIMR Berghofer Medical Research Institute. Marizomib was purchased from Cayman Chemicals (Cat #: 10007311). STF-31 was purchased from Selleck Chemicals (Cat #: S7931). The list of antibodies and RT-qPCR primers used in this study is provided in Table S1 and S2, respectively.
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