A rat insulinoma cell line (RINm5f beta) was purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). Cells from passages 30–43 were used. The cells were grown at 37 °C under a humidified 5% CO2 atmosphere in Roswell Park Memorial Institute (RPMI-1640, Sigma, St. Louis, MO, USA) medium supplemented with 10% fetal bovine serum (FBS, Sigma-Aldrich, St. Louis, MO, USA) and 1% antibiotic-antimycotic (ABAM, Gibco-Invitrogen, Grand Island, NY, USA). RINm5f beta cells were trypsinized using 0.05% trypsin ethylene diamine tetraacetic acid (EDTA, Sigma-Aldrich, St. Louis, MO, USA) at 80% confluence and loaded in a 96-well plate (Dutscher, Issy-les-Moulineaux, France) or 6-well plate (Dutscher) at a concentration of 105 cells/200 µL and 106 cells/mL, respectively. The medium was changed every two days. Twelve hours before starting the treatment, the cells were starved and incubated with different concentrations of plant extracts (20, 50, 80, 100, 150, 200, and 300 µg/mL for the aqueous jojoba seed extract, and 10, 20, 40, 80, and 150 µg/mL for simmondsin) for 24 h to study the toxicity on RINm5F beta cell lines (Figure S1A ). To validate the antioxidant properties of the extracts, cells were incubated with selected concentrations of plant extracts for 24 h before inducing an oxidative stress by 250 mM of fructose for 24 h (Figure S1B ).
96 well plate
Manufactured by Dutscher
Sourced in France
96-well plates are a type of laboratory equipment used for various experimental and testing purposes. They consist of a grid of 96 individual wells, typically arranged in a 8x12 format. These plates provide a standardized and efficient platform for performing multiple small-scale experiments or assays simultaneously.
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Lab products found in correlation
Glomax multi microplate multimode reader, by Promega (3 mentions)
Insulin from bovine pancreas, by Merck Group (2 mentions)
Fetal bovine calf serum, by GE Healthcare (2 mentions)
3 isobutyl 1 methylxanthine, by Merck Group (2 mentions)
Penicillin streptomycin, by GE Healthcare (2 mentions)
Adipored assay reagent, by Lonza (2 mentions)
6 well plate, by Dutscher (1 mentions)
Fetal bovine serum (fbs), by Merck Group (1 mentions)
Rpmi 1640, by Merck Group (1 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (1 mentions)
Trypsin edta, by Thermo Fisher Scientific (1 mentions)
Ensight, by PerkinElmer (1 mentions)
7 protocols using 96 well plate
1
Evaluating Antioxidant Effects of Plant Extracts
2
Cyclic AMP-Responsive Luciferase Assay in Mouse Leydig Cells
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The MLTC-1 (mouse Leydig tumor cell–1) was obtained from the American Tissue and Cell Collection (ATCC, LGC Standards, Molsheim, France). Cell culture, plasmids and transfections were carried out according to the method described in our previous work [23 (link),30 (link)]. Cells were cultured at 37 °C and 5% CO2 in RPMI-1640 medium (Gibco, Invitrogen, Saint-Herblain, France) supplemented with 10% fetal bovine serum, 50 µg/mL gentamicin, 10 units of penicillin/mL and 10 µg/mL streptomycin. Cells were transfected with pGlosensor-22F cyclic AMP plasmid using X-tremeGENE HP DNA transfection reagent. DNA (100 ng plasmid per well) and X-tremeGENE HP DNA transfection reagent (0.3 µL per well) were mixed together with serum-free RPMI medium and incubated at room temperature for 30 min. This plasmid consists in firefly luciferase sequence fused to that of the protein kinase A cyclic AMP-binding domain in a way that allows control of its enzymatic activity by cyclic AMP. And then the cells were sub-cultured at a density of 100,000 cells/well in 96-well plate (Dutscher, Brumath, France) overnight at 37 °C under 5% CO2 before use of the cells in the assays.
3
Differentiation of 3T3-L1 Adipocytes
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For differentiation of murine 3T3-L1 cultures to adipocytes, cells were seeded into 96-well plates (Dutscher Scientific, Brentwood, UK) at a density of 20 000 cells per well in 100 µl maintenance medium. Following a 2-day stabilization period, they were switched to differentiation medium consisting of DMEM, 2 mM glutamine, 10 µg/ml penicillin/streptomycin, 10% fetal bovine calf serum (GE Healthcare Life Sciences, Buckinghamshire, UK), 500 μM 3-isobutyl-1-methylxanthine (Sigma-Aldrich Ltd) and 100 nM insulin from bovine pancreas (Sigma-Aldrich Ltd). After a further 2 days of culture, the medium was refreshed to start the incubation with various concentrations of the test substances. Dexamethasone was used as a positive control. Media were replenished every 2 days for a further 6 days. Lipid accumulation was visualized on day 8 using fluorescent Nile Red staining in accordance with the manufacturer’s instructions (AdipoRed Assay Reagent, Lonza Walkersville Inc, USA) and quantified using a microplate reader (GloMax Multi Microplate Multimode Reader, Promega, Madison). The fluorescence was measured with a filter giving an excitation at 490 nm and emission at 510–570 nm. The adipogenic effect was expressed as a fold change in emission signal intensity between untreated differentiated and treated differentiated 3T3-L1 cells.
4
Quantifying 3T3-L1 Cell Viability via MTT Assay
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Cell viability was assessed using a colorimetric 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay, which indirectly measures cell number by testing for activity of mitochondrial succinate dehydrogenase. The MTT assay has also been proven to be a reliable measure of toxicity (Mosmann, 1983 (link)). The 3T3-L1 cells were seeded into 96-well plates (Dutscher Scientific) and differentiated as described above for 8 days with the indicated treatments. The MTT assay was performed as follows. Cells were incubated with 100 µl of MTT solution (1 mg/ml) for 2 h and the test terminated by lysing the cells by adding 100 µl DMSO. As a measure of cell number, the optical density of the cell lysate was determined at 570 nm using a GloMax Multi Microplate Multimode Reader (Promega). The number of cells was directly proportional to the intensity of the signal. Cell viability was expressed as a percentage of the control, untreated samples.
5
Trypsinization for HepG2 Cell Seeding
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After washing
with sterile phosphate buffer saline (PBS), the cells were detached
by trypsinization (0.05% trypsin/EDTA from Gibco). Twenty-four hours
before exposure, HepG2 cells were seeded into 96-well plates (Dutscher,
France) in 200 μL of complete culture medium at a final concentration
of 1.104 cells per well for resazurin assay.
with sterile phosphate buffer saline (PBS), the cells were detached
by trypsinization (0.05% trypsin/EDTA from Gibco). Twenty-four hours
before exposure, HepG2 cells were seeded into 96-well plates (Dutscher,
France) in 200 μL of complete culture medium at a final concentration
of 1.104 cells per well for resazurin assay.
6
Differentiation of 3T3-L1 Cells to Adipocytes
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For differentiation of 3T3‐L1 cultures to adipocytes, cells were seeded into 96‐well plates (Dutscher Scientific) at a density of 20 000 cells per well in 100 μl maintenance medium. Following a 2 day stabilization period, they were switched to differentiation medium consisting of DMEM supplemented with 2 mm glutamine, 10 μg ml−1 penicillin/streptomycin, 10% fetal bovine calf serum (GE Healthcare Life Sciences), 500 μm 3isobutyl1methylxanthine (Sigma‐Aldrich Ltd) and 100 nm insulin from bovine pancreas (Sigma‐Aldrich Ltd). After a further 2 day incubation, the medium was refreshed but lacked insulin and contained various concentrations of the test neonicotinoids. Treatment with dexamethasone acted as a positive control. Media were replenished every 2 days for a further 6 days. Lipid accumulation on day 8 following induction of differentiation was visualized using fluorescent Nile Red staining in accordance with the manufacturer's instructions (AdipoRed™ Assay Reagent; Lonza, Walkersville, MD, USA) and quantified using a microplate reader (GloMax® Multi Microplate Multimode Reader; Promega, Madison, WI, USA).
7
Cytotoxicity of Iron Oxide Nanoparticles
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The cytotoxicities
of Fe3O4-PEG-DOTA/pHLIP NPs and Fe3O4-PEG-DOTA NPs were evaluated by the MTT viability assay.
4T1 cells were inoculated into 96-well plates (Dutscher, France) with
a density of 8000 per well in 200 μL of complete culture medium.
After adherence, Fe3O4-PEG-DOTA/pHLIP NPs or
Fe3O4-PEG-DOTA NPs at different Fe concentrations
(0–300 μg·mL–1) were added and
incubated for 24 h at 37 °C and 5% CO2. After adding
20 μL of the CCK-8 reagent to each well, incubation was continued
for another 1 h in the cell culture incubator. The absorbance (OD)
at 450 nm in each well was measured using a PerkinElmer EnSight (PerkinElmer,
Shanghai). The mean and standard deviation for the triplicate wells
were reported.
of Fe3O4-PEG-DOTA/pHLIP NPs and Fe3O4-PEG-DOTA NPs were evaluated by the MTT viability assay.
4T1 cells were inoculated into 96-well plates (Dutscher, France) with
a density of 8000 per well in 200 μL of complete culture medium.
After adherence, Fe3O4-PEG-DOTA/pHLIP NPs or
Fe3O4-PEG-DOTA NPs at different Fe concentrations
(0–300 μg·mL–1) were added and
incubated for 24 h at 37 °C and 5% CO2. After adding
20 μL of the CCK-8 reagent to each well, incubation was continued
for another 1 h in the cell culture incubator. The absorbance (OD)
at 450 nm in each well was measured using a PerkinElmer EnSight (PerkinElmer,
Shanghai). The mean and standard deviation for the triplicate wells
were reported.
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