sorafenib tosylate (Cat. No. S-8502) was purchased from LC Laboratories, dissolved in DMSO at 10 mM aliquots, and stored at −20°C. Tumor organoids were plated at a density of 5 × 103 cells in 15 μL BME2 droplets in order to form organoids. At day 6, sorafenib was added to the medium, and cell viability was measured after 6 days using CellTiter-Glo 3D reagent (Promega). Luminescence was measured on a Synergy H1 Multi-Mode Reader (BioTek Instruments). Results were normalized to vehicle (=100% DMSO). Curve fitting was performed using Prism (GraphPad) software and the nonlinear regression equation. All experiments were performed at least two times in duplicate. Results are shown as mean ± SEM.
Celltiter glo 3d reagent
Manufactured by Promega
Sourced in United States, Germany, Italy, France, Ireland
CellTiter-Glo 3D reagent is a luminescent cell viability assay that quantifies ATP, which indicates the presence of metabolically active cells. The reagent is designed for use with 3D cell culture models.
Automatically generated - may contain errors
Lab products found in correlation
Enspire, by PerkinElmer (4 mentions)
Geltrex ldev free reduced growth factor basement membrane matrix, by Thermo Fisher Scientific (3 mentions)
Fluostar optima microplate reader, by BMG Labtech (3 mentions)
Matrigel, by Corning (3 mentions)
Prism 6, by GraphPad (3 mentions)
Bca kit, by Thermo Fisher Scientific (3 mentions)
Luminoskan ascent, by Thermo Fisher Scientific (3 mentions)
Synergy h1 multi mode reader, by Agilent Technologies (2 mentions)
Ref3603, by Corning (2 mentions)
Gen5 microplate reader, by Agilent Technologies (2 mentions)
Glomax discover system, by Promega (2 mentions)
Varioskan flash, by Thermo Fisher Scientific (2 mentions)
Round bottomed ultra low attachment 96 well plates, by Corning (2 mentions)
Dispase, by Thermo Fisher Scientific (2 mentions)
Spectramax microplate reader, by Molecular Devices (2 mentions)
Ultra low attachment 96 well plate, by Corning (2 mentions)
Adenosine 5 triphosphate disodium salt hydrate, by Thermo Fisher Scientific (2 mentions)
Synergy ht, by Agilent Technologies (2 mentions)
Nicotinamide, by Merck Group (2 mentions)
Celltiter 96 aqueous one solution reagent, by Promega (2 mentions)
101 protocols using celltiter glo 3d reagent
1
Sorafenib Cytotoxicity in Tumor Organoids
2
Spheroid Viability Quantification
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Spheroids were gently pipetted from agarose gels. Individual spheroids in 100μL complete cortical media were transferred to an opaque-walled, clear bottom 96-well plate, using a 200μL pipette with the tip cut off to accommodate the spheroid. 100μL of CellTiter-Glo 3D Reagent (Promega Life Sciences) was added to each well. The plate was wrapped in aluminum foil and placed on a shaker for 10 minutes to induce lysis. The luminescence was measured using a Cytation5 (Biotek).
3
Assessing Pt(IV)-M13 Cytotoxicity
4
Radiosensitivity of Carcinoma Organoids
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Tumoroids derived from carcinoma (MEC and AdCC) were harvested and subcultured in 48-well plates as described above. Then, 3 days after seeding, the organoids were irradiated using the X-Rad320 (Precision X-Ray, North Branford, CT, USA). Separate culture plates were used for different radiation doses ranging from 0 to 10 Gy. Media were changed after radiation, and organoids were cultured for another 4 days. Cell viability was measured via the evaluation of ATP levels using the CellTiter-Glo 3D reagent (Promega), according to the manufacturer’s protocol. The results were normalized to baseline (0%, staurosporin 1 μM) and positive (100%, 0 Gy) controls.
5
Adenoma Organoid Cell Viability
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The adenoma organoids transfected with PLOD3-siRNA were used to detect cell viability. The CellTiter-Glo® 3D Reagent (Promega, G9681) was used to dissolve the matrigel. The adenoma organoid suspension was equally distributed to opaque-walled multiwell plates (Corning, REF3603). The volume of CellTiter-Glo® 3D Reagent equal to the volume of cell culture medium was added to each well and then the content was incubated for 30 min at room temperature. Cell viability was calculated based on ATP content of each well.
6
Organoid Viability Assay for Drug Testing
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Viability of the tumor and normal organoids following drug treatment was performed as previously described [23 (link)]; see additional details in the Supplementary Methods . Briefly, organoids were collected with an organoid harvesting solution (Cultrex, R&D Systems, Minneapolis, MN, USA) and digested to single cells with TrypLE Express (Gibco, Waltham, MA, USA). Single cells were suspended in AdDE+++ medium with 10% Matrigel and seeded at a density of 10,000 cells/well in an ultra-low attachment 96-well U-bottom white plate. After 1–4 days, the organoids were exposed to carboplatin (1 µM), cisplatin (1 µM), paclitaxel (10 nM), bevacizumab (1 µM), gemcitabine (100 nM), topotecan (100 nM) or different combinations for 72 h at 37 °C. At the end of the incubation, an equal volume of the CellTiter-Glo 3D reagent (Promega, Madison, WI, USA) was added to each well and incubated for 25 min at room temperature. Luminescence, reflective of cell viability, was measured using a Gen5 Microplate Reader (BioTek, Winooski, VT, USA). All the tests were conducted in triplicate and the data were normalized to untreated controls (set at 100% viability).
7
Nanoparticle Synthesis and Characterization
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To prepare nanoparticles, poly-L-arginine hydrochloride (M w = 5800 Da) was provided by Polypeptide Therapeutic Solutions S. L. (Valencia, Spain) and low molecular weight hyaluronic acid (M w ≈ 20 kDa) was purchased from Lifecore® Biomedical (Chaska, USA). AmberLite® IRA 900 Cl ion exchange resin, dichloro(1,2-diaminocyclohexane)platinum(II) (DACHPt-Cl 2 , M w = 380.17 Da) and silver nitrate (AgNO 3 , M w = 169.97 Da) were purchased from Sigma-Aldrich® (Saint-Quentin-Fallavier, France). Sodium hydroxide (NaOH) was purchased from Acros Organics (Thermo Fisher Scientific, Illkirch, France). Oxaliplatin solution (Hospira®, Meudon, France) was a kind gift from the Hospital of Lyon, France. Deionized water (Milli-Q water) was obtained from a Milli-Q plus system (Merck-Millipore, Darmstadt, Germany). A CellTiter Glo® 3D reagent was purchased from Promega Corporation (Madison, USA). To prepare microwells, agarose standard (ROTI®Garose) was purchased from Carl Roth® (Karlsruhe, Germany) and the Norland Optical Adhesive 81 (NOA) glue was provided by Norland Products Inc. (Cranbury, USA). For coverslip silanisation, 3-aminopropyltriethoxysilane 99% (APTS) came from AcroSeal® (Heysham, United Kingdom) and glacial acetic acid was supplied by VWR PROLABO® Chemicals (Fontenay-sous-Bois, France).
8
Tumor Organoid Cytotoxicity Assay
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Tumor organoids were plated as single cells in a 96-well plate at a density of 1 × 104 cells in 10 μL Matrigel droplets. Before treatment, cells were allowed to recover and form organoids for 2 to 3 days. At day 3, 6-MP at a final concentration of 2.5 µM or 5-FU at concentrations of 1.25, 2.5 and 5 µM or in combination was added to the medium, and cell viability was assessed after 5 day using CellTiter-Glo 3D reagent (#G9682, Promega). Luminescence was measured on Varioskan Microplate Reader (ThermoFisher Scientific). Results were normalized to DMSO control. All experiments were performed twice in quadruplicate.
9
Spheroid Assay for OVCA420 Cells
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OVCA420* cells stably expressing inducible TIGAR shRNAs (TRIPZ inducible lentiviral shRNAs were purchased from Dharmacon, US) were established by selection with puromycin after viral transduction. Stable cells were treated with 1 µg/mL doxycycline for 72 h to induce expression of shRNAs. Then 3000 cells/well were seeded into 96-well plates (plates were pre-coated with 100 µL 1.5% (wt/vol) agarose) and treated with vehicle or different concentrations of olaparib for 10 days. At the end of the experiment, pictures of spheroid were taken under a microscope and cell viability was measured with CellTiter-Glo 3D reagent (Promega Corporation).
10
Cell Viability Assay with CellTiter-Glo3D
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MDA-MB-231 and U-251 MG cells were seeded at a density of 3.0 × 103 cells/90 μL per well in 96-well plates (3596; Corning, NY, USA) and treated with the test compounds (10 μL per well) 24 h after seeding. After 3 d, CellTiter-Glo® 3D Reagent (Promega, Madison, WI, USA) was added to each well at an equivalent volume to the cell culture medium in the well, mixed by shaking for 5 min at room temperature, and incubated for 25 min at 37 °C under 5% CO2. The 100 μL supernatants were then transferred in technical replicates to 96-well white plates (136101; Thermo Fisher Scientific), and their luminescence was measured using a luminometer (GloMax® Discover System; Promega).
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