Immature and mature thyroid cell spheroids were collected and seeded into white 96-well plates (PerkinElmer, Waltham, MA, USA) treated with antiadherence rinsing solution (StemCell Technologies) and incubated overnight. Then, the spheroids were incubated with RealTime-GloTM Assay Reagent (Promega) and exposed to increasing concentrations of menadione. Luminescence was measured at the indicated times using a Victor3 microplate reader. EC50 values (50% decrease in viable cells) were calculated using Prism 8.0 software.
White 96 well plate
Manufactured by PerkinElmer
Sourced in United States
The White 96-well plates are a laboratory equipment product designed for use in various assays and experiments. The plates feature a sturdy, white polystyrene construction and contain 96 individual wells, each with a flat bottom. The plates are compatible with standard laboratory equipment and are suitable for a range of applications.
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Lab products found in correlation
Atplite 1 step kit, by PerkinElmer (5 mentions)
Envision 2104 multilabel reader, by PerkinElmer (3 mentions)
Hek293t, by Merck Group (2 mentions)
Dmem glutamax, by Thermo Fisher Scientific (2 mentions)
Jetpei reagent, by Polyplus Transfection (2 mentions)
Poly l lysine (pll), by Merck Group (2 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions)
Envision multilabel plate reader, by PerkinElmer (2 mentions)
Dual luciferase assay kit, by Promega (2 mentions)
Dual glo luciferase assay, by Promega (2 mentions)
Envision plate reader, by PerkinElmer (2 mentions)
Microscint 0, by PerkinElmer (2 mentions)
Methyl 3h thymidine, by PerkinElmer (2 mentions)
Topcount, by PerkinElmer (2 mentions)
Anti adherence rinsing solution, by STEMCELL (1 mentions)
E1483, by Promega (1 mentions)
Enspire multimode plate reader, by PerkinElmer (1 mentions)
Quercetin, by Merck Group (1 mentions)
Opti mem media, by Thermo Fisher Scientific (1 mentions)
Minimum essential media, by Merck Group (1 mentions)
36 protocols using white 96 well plate
1
Thyroid Cell Spheroid Viability Assay
2
Ginger Extract and Quercetin Effects on Reporter Cells
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BJ and HaCaT reporter cells were seeded in white 96-well plates (PerkinElmer) at concentration of 2 × 105 in 100 μL of Minimum Essential Media (Sigma-Aldrich) supplemented by 1% nonessential amino acids (NEAA), 1 mM sodium pyruvate, 10% FCS, streptomycin (100 μg/mL), and penicillin (100 IU/mL) for 24 h at 37°C and 5% CO2. The media was replaced by Opti-MEM media (Gibco) containing 0.5% FBS and 1% NEAA and cells were treated by 40 μg/mL of ginger extract or 30 μM quercetin (Sigma-Aldrich) while the control wells contained media with corresponding concentration of the solvent 0.05% dimethyl sulfoxide (DMSO) as described by Bak et al. [17 (link)]. Following 10 h treatment, the cells were lysed by 20 μL of cell culture lysis buffer (E153A, Promega) and 100 μL of luciferase assay substrate (E1483, Promega) was added. Luminescence was measured by EnSpire multimode plate reader (PerkinElmer). The values from luminescence assays were normalized by values acquired from MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assays with the cells that were prepared simultaneously with the same treatment conditions as described by Yamazaki et al. [18 (link)]. All the assays were performed three times with five technical replicates for each treatment.
3
Evaluating Glioma Cell Viability
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Optimized number of U87MG, A172, U118, T98G and E98 cells (3 × 103 to 5 × 103) were seeded in white 96-well plates (Perkin Elmer) and allowed to attach. After 24 h, cells were treated with determined concentration of chemical compounds for 24–72 h. Cell viability was measured using a CellTitre Glo assay (Promega) according the manufacturer’s instructions. Luminescence was read with BioTek Synergy H1 plate reader (BioTek, Country). Bioluminescence was normalized and presented as a per cent of the control.
Cell viability for glioma stem cells was seeded into 96-well plates 5 × 103 cells/well and allowed to attach overnight. Next day medium was changed into a medium containing drugs. After 72 h of treatment medium was changed into fresh NSC+/+ containing 10% of Alamar blue solution (10099022, Fisher Scientific). Fluorescent signal was measured after 210 min of incubation in 37°C using Wallac Victor 1420 plate reader (Perkin Elmer). Fluorescence was normalized and presented as a per cent of the control.
Cell viability for glioma stem cells was seeded into 96-well plates 5 × 103 cells/well and allowed to attach overnight. Next day medium was changed into a medium containing drugs. After 72 h of treatment medium was changed into fresh NSC+/+ containing 10% of Alamar blue solution (10099022, Fisher Scientific). Fluorescent signal was measured after 210 min of incubation in 37°C using Wallac Victor 1420 plate reader (Perkin Elmer). Fluorescence was normalized and presented as a per cent of the control.
4
HEK293T and SH-SY5Y Cell Culture and Transfection
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HEK293T (Sigma-Aldrich; RRID: CVCL 0063; authenticated by the provider) were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM) Glutamax (Invitrogen) supplemented with 4.5 g/L glucose, 10% fetal bovine serum, 100 U/mL penicillin, and 0.1 mg/mL streptomycin, at 37 °C (95% O2, 5% CO2). SH-SY5Y cells were maintained in DMEM:F12 Medium (Invitrogen) supplemented with 10% fetal bovine serum, 100 U/mL penicillin, and 0.1 mg/mL streptomycin, at 37 °C (95% O2, 5% CO2). Cell lines were checked regularly for mycoplasma contamination. Cells were transfected with equal amounts of LgBit and SmBit construct (250 ng each per well in 6-well plates; or 125 ng each per well in 12-well plates) or 250 ng (6-well plates) of the intramolecular sensor. For the saturation curves, specific amounts of each construct were used, as indicated. Transfections were performed using jetPEI reagent according to the supplier’s instructions (Polyplus-transfection, New York, NY, USA). One day after transfection, cells were plated into white 96-well plates (Perkin Elmer Life Sciences, Waltham, MA) precoated with 10 μg/ml poly-L-lysine (Sigma-Aldrich), and the Nluc complementation assay was performed 48 hr post-transfection.
5
Wnt Signaling in HEK293T Cells
6
Reactive Oxygen Species Assay
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Two thousand five hundred cells/well were plated in white 96-well plates (PerkinElmer, Waltham, MA) and treated the following day with DMSO, OT-82, NMN, or the combination. N-acetylcysteine (Sigma) was pH-adjusted to 7.8 in water and added to cells. Cells were processed according to the ROS-Glo assay protocol (Promega). Luciferase activity was measured with a Molecular Device SpectraMax M3 plate reader. Each experiment was performed at least two times with three technical replicates.
7
Luciferase Reporter Assay in HEK293 Cells
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HEK293 cells were seeded on white 96-well plates (Perkin-Elmer) at a density of 2.5 × 104 cells per well. Twenty-four hours later, cells were transfected with 25 ng of firefly luciferase reporter plasmid, 5 ng of the internal control reporter plasmid pRL-TK (Promega), and 25 to 50 ng of the pcDNA3-1(−) plasmid containing a cDNA of interest, or the empty plasmid, using Lipofectamine 2000. Forty-eight hours posttransfection, cells were lysed and assayed with the Dual-Glo luciferase system (Promega), according to the manufacturer's instructions. The luciferase activity of each well was measured using the Veritas microplate luminometer (Turner Biosystems). For each well, the firefly luciferase activity was normalized to the renilla luciferase reading and plotted as fold induction, compared to the empty pcDNA3.1(−) vector. Each condition was performed in quintuplicate.
8
SARS-CoV-2 Pseudovirus Neutralization Assay
9
Wnt Signaling Pathway Reporter Assay
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Wildtype human embryonic kidney 293T cells maintained in Dulbecco's modified Eagle's medium supplemented with 10% (v/v) fetal bovine serum (Gemini) were seeded on white 96-well plates (PerkinElmer) at 7500 cells/well. About 24 h later, cells were transfected with receptor vectors (1 ng each/well), Super8xTOPFlash (Addgene plasmid no.: 12456; 80 ng/well), and LacZ under a cytomegalovirus promoter (20 ng/well) using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions. About 16 to 20 h post-transfection, media were replaced with L-cell control or Wnt3a-conditioned media. Cells were lysed 22 to 26 h later, and the Dual-Light system (Invitrogen) was used according to the manufacturer's instructions to quantify luciferase and β-galactosidase activity using a plate reader (BioTek Synergy2).
10
Measuring GPCR-Effector Interactions
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For arrestin recruitment assays, FLIP-IN HEK-393 cells stably expressing Arr3-NLuc were transiently transfected with SYFP2-labelled receptor as previously described. For Gαs recruitment assays, FLIP-IN HEK-393 cells stably expressing either the GLP-1R-NLuc or GCGR-NLuc were transiently transfected with NES-Venus-Gαs. 48 h post-transfection cells were detached and washed with Hank’s Balance Salt Solution (HBSS). Cells were re-suspended in HBBS and plated on to white 96-well plates (PerkinElmer) in suspension at a density of 180,000 cells/well. Cells were incubated with agonist for 15 min and BRET measurements were taken using a Victor X4 (PerkinElmer) plate reader immediately after the addition of coelenterzine h (final conc. 5 µM). NLuc emission was measured through a 460/40 nm filter and the resulting SYFP2 emission was read through a 535/25 nm filter.
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