The cell lysate was thawed at room temperature. Stock solutions of the isoflavonoids and positive controls were dissolved in DMSO and 10-fold serially diluted in the cell lysate to the final concentration with 1% DMSO. Twenty microliters of each isoflavonoid in the cell lysate were transferred to 96-well plates in triplicate and incubated for 30 min at room temperature. Then, 30 µL Luciferase Assay Reagent II (contained in the Dual-Luciferase Reporter Assay System; Promega, Madison, WI, USA) was added to each well, and the FLuc luminescence was measured using a microplate reader (Synergy 4 hybrid; BioTek, Winooski, VT, USA). The addition of 30 µL of Stop & Glo Reagent (contained in the Dual-Luciferase Reporter Assay System) followed, and the RLuc luminescence was recorded using the microplate reader (Synergy 4 hybrid; BioTek, Winooski, VT, USA).
Synergy 4 hybrid
Manufactured by Agilent Technologies
Sourced in United States
The Synergy 4 Hybrid is a multi-mode microplate reader capable of performing absorbance, fluorescence, and luminescence detection. It features a combination of filter-based and monochromator-based optics to provide flexibility in experimental setup and data collection.
Automatically generated - may contain errors
Lab products found in correlation
Lsm 510 meta, by Zeiss (1 mentions)
Nile red staining, by Merck Group (1 mentions)
Black 384 well plate, by Greiner (1 mentions)
Glycolysis cell based assay kit, by Cayman Chemical (1 mentions)
Mitoxpress xtra hs, by Cayman Chemical (1 mentions)
Atp bioluminescence assay kit cls 2, by Roche (1 mentions)
Mouse tnf α duoset elisa development kit, by R&D Systems (1 mentions)
Mouse il 1β duoset kit, by R&D Systems (1 mentions)
Resazurin, by Merck Group (1 mentions)
Resorufin, by Agilent Technologies (1 mentions)
Methanol, by Merck Group (1 mentions)
Dual luciferase reporter assay system, by Agilent Technologies (1 mentions)
Luciferase assay reagent 2, by Promega (1 mentions)
Dual luciferase reporter assay system, by Promega (1 mentions)
8 protocols using synergy 4 hybrid
1
Quantification of Isoflavonoid-Mediated Luciferase Activity
2
Fura-2-AM Calcium Imaging Assay
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Cells (1 × 106 cells ml−1) were incubated with 1 μM Fura-2-AM for 30 min at 37 °C in extracellular calcium buffer (ECB, 130 mM NaCl, 5 mM KCl, 1.5 mM CaCl2, 1 mM MgCl2, 25 mM Hepes, pH 7.5, 1 mg ml−1 BSA, and 5 mM glucose) in dark, after which they were collected and resuspended in ECB for an additional incubation at 25 °C for 30 min to permit dye de-esterification. Cells were then collected and resuspended to 2 × 106 cells ml−1 in ECB. Cells in 100 μl ECB were transferred to a 96-well black-wall cell culture plates and fluorescence was continuously recorded at 25 °C (alternating 340 and 380 nm excitation, 510 nm emission) in a Microplate Reader (Synergy 4 Hybrid, BioTek).
3
Microalgal Lipid Quantification and Fatty Acid Analysis
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Cellular neutral lipid content was determined by using Nile Red staining (Sigma, USA) according to Yang et al. [11 (link)]. Staining was performed in a 96-well microtitre plate in triplicates and fluorescence was recorded using Synergy 4 Hybrid (BioTek, USA) at a wavelength of 530 nm excitation and 580 nm emission. For confocal microscopic observation of microalgal morphology, cells were stained with Nile red and lucifugally incubated for 10 min at room temperature. Stained cells were observed under a laser-scanning confocal microscope Zeiss LSM510meta (Zeiss, Germany) with an excitation wavelength of 488 nm and an emission wavelength of 505–550 nm.
Total lipids from the microalgae were extracted as per the protocol reported by Bligh and Dyer [27 (link)]. Cells were subjected to nitrogen deprivation to validate the lipid accumulating capability as previously described [9 (link)]. Fatty acid composition of the total lipids was analyzed as fatty acid methyl esters (FAMEs) by using gas chromatography–mass spectrometry (GC–MS) according to the protocol reported previously [11 (link)].
Total lipids from the microalgae were extracted as per the protocol reported by Bligh and Dyer [27 (link)]. Cells were subjected to nitrogen deprivation to validate the lipid accumulating capability as previously described [9 (link)]. Fatty acid composition of the total lipids was analyzed as fatty acid methyl esters (FAMEs) by using gas chromatography–mass spectrometry (GC–MS) according to the protocol reported previously [11 (link)].
4
Quantifying Cellular IP3 Dynamics
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IP3 was measured using the HitHunter IP3 Fluorescence Polarization Assay Kits (DiscoverRx Tech, Fremont, CA, USA). Briefly, 2 × 104 cells in black 384-well plates (Greiner, Germany) were treated with anti-CD3 for the designated times, and the cellular reaction was terminated by adding 0.2 N perchloric acid. The plate was shaken at 650 r.p.m. for 15 min. The IP3 tracer was subsequently added to each well, and the IP3 binding protein was added to the plate. The polarized fluorescence from the IP3 tracer was read on a Microplate Reader (Synergy 4 Hybrid, BioTek, ). The IP3 concentration was calculated from the IP3 standard curve.
5
Oxygen Consumption and Metabolic Assays
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Oxygen consumption was assessed using MitoXpress-Xtra-HS (Cayman Chemical, Ann Arbor, MI, USA), a porphyrin-based phosphorescent oxygen-sensitive probe. Before assay, cells were transferred into fresh culture medium containing 1% FBS. Overall, 10 μl of probe was added and the cells were equilibrated at 37 °C. The assay was read using a Microplate Reader (Synergy 4 Hybrid, BioTek, Winooski, VT, USA). The maximal rate of oxygen consumption is proportional to the change in probe fluorescence during the linear phase of the assay. The lactic acid levels and ATP levels were determined by glycolysis cell-based assay kit (Cayman Chemical) and ATP Bioluminescence Assay Kit CLS II (Roche, Basel, Switzerland) according to the manufacturer's instructions.
6
Quantifying Cytokine Secretion in Amyloid-treated Cells
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BV2 cells (105 cells/well) were incubated in a 24-well plate, and media were collected after β-amyloid treatment. Secreted TNF-α in media was measured using a mouse TNF-α DuoSet ELISA Development kit (R&D Systems Inc., Minneapolis, MN, USA) and mouse IL-1β DuoSet kit (R&D Systems Inc.). Optical density was detected using a Synergy 4 Hybrid microplate reader (BioTek, Winooski, VT, USA), and cytokine level was deducted from the absorbance value by extrapolation from a standard curve generated in parallel.
7
Evaluating Metabolic Activity in Cell Cultures
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To evaluate the metabolic activity of each cell line or coculture, 100 µL of cells/well were seeded in black 96well plates and incubated for 24 h. An amount of 10 µL of 400 µM resazurin (Sigma Aldrich) dissolved in PBS was added to the cells and further incubated for 3 h. Resorufin fluorescence was measured at λ ex /λ em = 530/590 nm using an automated plate reader (Synergy 4 Hybrid; BioTek, Winooski, VT, USA). Two independent experiments were performed in technical duplicates. The effect of immunosuppressive drugs on the metabolic activity of cocultures was also investigated. An amount of 100 µL of the coculture/well was seeded into black 96-well plates. On a separate plate, the bisphenols were first appropriately diluted in DMSO, then diluted in the medium and added to the cells. Concentrations between 1 nM and 50 µM were tested. As control samples, the cells were exposed to a vehicle control (0.5% DMSO). After 24 h of incubation, 10 µL of 400 µM resazurin was added and Resorufin fluorescence was recorded after 3 h as described above. Three independent experiments were performed with technical duplicates.
8
Quantifying Chlorophyll and Carotenoids
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To determine concentrations of chlorophyll-a and carotenoids, samples were collected according to the sampling schedule, placed on ice, centrifuged, resuspended in 99.8% methanol (Sigma Aldrich Inc.), and stored in the dark for a minimum of 24 h. Aliquots of each extract were transferred into wells of a 96-well plate and measured for optical density using a microplate spectrophotometer (BioTek Synergy 4-Hybrid). Chlorophyll-a and total carotenoids were calculated using the method for methanol extraction described by [52 ]. Chlorophyll and carotenoid concentrations per cell (ng/cell) were calculated using the following equation:
where, X = pigment concentration per cell in ng/cell, C = pigment concentration in µg/mL of extract, and N = number of cells per mL.
where, X = pigment concentration per cell in ng/cell, C = pigment concentration in µg/mL of extract, and N = number of cells per mL.
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