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7 protocols using ink128

1

Plate Colony and Soft-Agar Assays with Kinase Inhibitors

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For plate colony assay, 500 to 1000 cells per well (cell line dependent) were seeded in six-well plates in 2 ml of media and resupplied with fresh media every week. After 2 to 3 weeks, cells were fixed with 10% (w/v) formaldehyde for 15 min and stained with 0.05% (w/v) crystal violet supplemented with 10% ethanol and 10% methanol for 20 min at room temperature. The plates were then washed with distilled water, air dried, and scanned using a Canon scanner. The cell colony numbers were then manually counted. Soft-agar assays were performed as previously described (19 (link)). When applicable, AKT inhibitors MK2206 (#S1078, Selleckchem) and GSK1411795 (#S7492, Selleck), and mTOR kinase inhibitors PP242 (#S2218, Selleckchem) and INK128 (#HY-13328, MedChemExpress) were added during setup and added with fresh media every week.
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2

Oxygen Tension Effects on UC-MSCs

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UC-MSCs were incubated under different O2 concentrations for 24 and 48 h: 5% O2 in a humidified incubator (5% CO2, 90% N2, 37 °C); 1% O2 in a hypoxia chamber (STEMCELL Technologies Inc., Cambridge, MA, USA) (5% CO2, 94% N2, 37 °C); <1% O2 in a BD GasPak EZ anaerobe gas-generating pouch system (Becton, Dickinson and Company, Sparks, MD, USA) at 37 °C [36 (link)]. For chemical hypoxia induction, culture media was exchanged by medium containing the hypoxia-mimicking agent cobalt (II) chloride hexahydrate (CoCl2.6H2O; Sigma-Aldrich, Burlington, MA, USA) [24 (link)] at several concentrations (10 µM, 50 µM, 100 µM, 200 µM, and 250 µM), after which the cells were incubated for 24 and 48 h (21% O2, 5% CO2, 37 °C). To induce a paused-like state, and as a control condition for mTOR dual inhibition, cells were treated with the mTOR pharmacological inhibitor INK-128 (100 nM, MedChem Express, Monmouth Junction, NJ, USA).
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3

Detailed Protocols for Molecular Biology Experiments

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All chemicals and reagents in this work were purchased and used without further treatment or purification. General laboratory reagents were purchased from Sigma-Aldrich (UK) unless otherwise stated. AZD2014 and INK128 were purchased from MedChem Express (Europe). Cell culture dishes (35 mm × 20 mm) were bought from MatTek (USA) and 96-well round plates from (Corning™ Falcon™). Propidium iodide was purchased from Sigma-Aldrich (UK). ER-Tracker™ Red and LysoTracker Red DND-99 was purchased from Thermofisher Scientific (UK). mDsRed-Rheb, EGFP-Rheb, EGFP-mTOR and EGFP-S6K1 DNA plasmids made as previously described [14 (link)] [45 (link)]. EGFP-raptor plasmid was a kind gift from Prof. Jacek Jaworski (Poland) and purified GFP was a kind gift from Prof. Cameron Naylon (Australia).
All cell culture reagents were purchased from Thermofisher Scientific (Gibco™).
Human Embryonic Kidney (HEK293) cells were purchased from ATCC (U.S.A.), certified as mycoplasma (contamination) free. Michigan Cancer Foundation-7 (MCF-7) breast cancer and Chinese Hamster Ovary (CHO) cells were initially purchased from ECACC (UK) and mycoplasma free.
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4

Evaluating Cell Proliferation with Crystal Violet Assay

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Cells were seeded at 5000 to 20,000 cells per well (cell line dependent) in 500 μl of media in 24-well plates. Cell cultures were stopped at predetermined time points, fixed with 10% (w/v) formaldehyde for 15 min, and stained with 0.05% (w/v) crystal violet in 10% ethanol and 10% methanol for 20 min at room temperature. After wash and air dry, 300 μl 10% acetic acid was added to each well, and absorbance was read at 570 nm. Dox was used for inducing gene overexpression (1 μg/ml) or knockdown (4 μg/ml). For proliferation assays on cells with siRNA-mediated knockdown of MAPK6, cells were first transfected with 10 nM siRNA. Forty-eight hours later, cells were plated in 24-well plates and used in proliferation assays. When applicable, mTOR kinase inhibitors PP242 (#S2218, Selleckchem) and INK128 (#HY-13328, MedChemExpress) were added to culture media the day after cell seeding.
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5

Quiescent ESCs via FAO or MYC Inhibition

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Naive ESCs were induced to achieve a stable quiescent state by inhibition FAO or MYC (FAOi or MYCi). To inhibit FAO, Etomoxir (SIGMA ALDRICH, #E1905, 100 µM) was added at the time of plating for 4 days5 (link). 10058-F4 (SIGMA ALDRICH, #475956) was used at a final concentration of 64 µM for 3 days to inhibit MYC6 (link). As a control, naive ESCs were treated with mTOR inhibitor (mTORi) INK128 (Medchem Express, HY13328, 200 nM) for 3 days as described7 (link). H2O and DMSO were added at the same final volume as FAOi, MYCi or mTORi. The induced qESCs and control cells were then harvested for RT-qPCR, metabolomics analysis and TSC differentiation.
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6

Optimized Preimplantation Embryo Culture

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Unless otherwise indicated, Swiss Webster females were mated to Swiss Webster males, or to C57BL/6 males homozygous for an Oct4/GFP transgene (B6.Cg-Tg(Pou5f1-GFP)1Scho)25 (link). Preimplantation embryos were harvested at indicated time-points after detection of the copulatory plug by flushing oviducts (E1.5-E2.5) or uteri (E3.5) of pregnant females using M2 medium (Zenith Biotech) supplemented with 2% BSA (Sigma). Subsequent embryo culture was performed in 4-well plates in 5% O2, 5% CO2 at 37°C in KSOMAA Evolve medium (Zenith Biotech) with 2% BSA and the following inhibitors, after optimization of concentrations: 200 nM INK128 (Medchem Express), 2.5 μM 10058-F4 (Sigma), 100 ng/ml Cycloheximide (Amresco), 50 μM Anacardic Acid (Sigma). Other mTor inhibitors [AZD2014, Everolimus and Rapamycin (Medchem Express) and RapaLink-1 (gift of Kevan Shokat)] and autophagy inhibitors chloroquine (Sigma) and SBI-0206965 (Medchem Express) were used at the indicated concentrations under same culture conditions.
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7

Optimized Preimplantation Embryo Culture

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Unless otherwise indicated, Swiss Webster females were mated to Swiss Webster males, or to C57BL/6 males homozygous for an Oct4/GFP transgene (B6.Cg-Tg(Pou5f1-GFP)1Scho)25 (link). Preimplantation embryos were harvested at indicated time-points after detection of the copulatory plug by flushing oviducts (E1.5-E2.5) or uteri (E3.5) of pregnant females using M2 medium (Zenith Biotech) supplemented with 2% BSA (Sigma). Subsequent embryo culture was performed in 4-well plates in 5% O2, 5% CO2 at 37°C in KSOMAA Evolve medium (Zenith Biotech) with 2% BSA and the following inhibitors, after optimization of concentrations: 200 nM INK128 (Medchem Express), 2.5 μM 10058-F4 (Sigma), 100 ng/ml Cycloheximide (Amresco), 50 μM Anacardic Acid (Sigma). Other mTor inhibitors [AZD2014, Everolimus and Rapamycin (Medchem Express) and RapaLink-1 (gift of Kevan Shokat)] and autophagy inhibitors chloroquine (Sigma) and SBI-0206965 (Medchem Express) were used at the indicated concentrations under same culture conditions.
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