Protein estimation was performed using bicinchoninic acid (BCA) Micro BCATM Protein assay kit (Thermo Fisher Scientific, Waltham, MA, USA) according to manufacturer’s instructions and the absorbances were read using SpectraMax 190 Absorbance Microplate Reader (Molecular Devices, San Jose, CA, USA).
Spectramax 190 absorbance microplate reader
Manufactured by Molecular Devices
Sourced in United States
The SpectraMax 190 Absorbance Microplate Reader is a lab equipment designed to measure the absorbance of samples in microplates. It is capable of reading 96-well microplates and can be used for various applications requiring absorbance measurements.
Automatically generated - may contain errors
Lab products found in correlation
Softmax pro computer software, by Molecular Devices (2 mentions)
Cell proliferation reagent wst 1, by Roche (2 mentions)
Protease inhibitor mixture tablet, by Roche (1 mentions)
Methyl thiazolyl tetrazolium (mtt), by Merck Group (1 mentions)
Maldi tof toftm 5800 system, by AB Sciex (1 mentions)
Celltiter 96 aqueous one solution reagent, by Promega (1 mentions)
Softmax pro software v5, by Molecular Devices (1 mentions)
Quantikine elisa kit, by R&D Systems (1 mentions)
Mtt kit, by Beyotime (1 mentions)
Softmax pro, by Molecular Devices (1 mentions)
Pahbah, by Merck Group (1 mentions)
96 well pcr plate, by Thermo Fisher Scientific (1 mentions)
Ptc 200 thermal cycler, by Bio-Rad (1 mentions)
Sodium hydroxide (naoh), by Merck Group (1 mentions)
Celltiter 96 aqueous non radioactive cell proliferation assay, by Promega (1 mentions)
Triton x 100 1, by Merck Group (1 mentions)
Human duo set kit, by R&D Systems (1 mentions)
Verikine human ifn beta elisa kit, by PBL Assay Science (1 mentions)
Softmax pro software, by Molecular Devices (1 mentions)
Automated cell counter, by Thermo Fisher Scientific (1 mentions)
21 protocols using spectramax 190 absorbance microplate reader
1
Protein Quantification using BCA Assay
2
Quantification of Ovotransferrin by ELISA
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Quantification of OTf by this test was performed using Chicken Ovotransferrin ELISA Kit (MyBioSource); 100 μL of serial dilutions of standard OTf in PBS was distributed in each well (6.75 ng/mL, 12.5 ng/mL, 25 ng/mL, 50 ng/mL, 100 ng/mL, and, resp., 200 ng/mL) and OTf samples were analyzed. The plate was incubated in dark place for 30 minutes at 25°C, covered. After incubation the plate was washed 4 times with washing buffer (Wash Buffer) diluted 20 times. To each well 100 μL of the peroxidase (HRP) labeled enzyme conjugate was added, diluted 100 times. The plate was incubated in the dark for 20 minutes at 24°C and to each well 100 mL of TMB was added and kept at the room temperature for 10 minutes. Next, 100 μL/well of stop solution was added and absorbance was read immediately at 450 nm (OD 450) using a SpectraMax 190 Absorbance Microplate Reader (Molecular Devices). OTf purified in serial dilutions of PBS was used to create a standard curve. This curve was used to estimate the OTf's total concentration in the samples.
3
SHP2 Phosphatase Activity Assay
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SHP2 activity was analyzed using a Human Active SHP-2 kit (R&D Systems Inc., Minneapolis, MN, USA). Briefly, cells were lysed in a lysis buffer ([50 mM HEPES, 0.1 mM EGTA, 0.1 mM EDTA, 120 mM NaCl, 0.5-% Nonidet-P40 [NP-40], pH 7.5 supplemented with fresh protease-inhibitor-mixture tablets (Roche Applied Science). The SHP2 proteins were then immunoprecipitated using active SHP2 immunoprecipitation beads (R&D Systems Inc.), and washed 3 times in the lysis buffer and 4 times in a phosphatase assay buffer (10 mM HEPES, 0.1 mM EGTA, 0.1 mM EDTA, 0.5-% BSA, 1 mM dithiothreitol [DTT], pH 7.5). The phosphatase reaction was initiated by incubating the immunocomplexes for 30 min at 37°C in the presence of tyrosine phosphatase substrate I, DADEY (PO3) LIPQQG, according to the manufacturer's instructions. Phosphatase activity was determined using a microplate reader (SpectraMax 190 Absorbance Microplate Reader; Molecular Devices) at 620 nm.
4
MTT Assay for Cell Proliferation
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3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl-tetrazolium bromide (MTT) (Sigma-Aldrich) assays were used to quantify cell proliferation rates as described previously [45 (link)]. Briefly, 4 x 103 cells were seeded in 96-well plates and cultured at 37 °C in the presence of 5 % CO2 until required for analysis at 24 h, 48 h and 72 h time points. On the day of analysis, the growth media was discarded and MTT (dissolved in 1X PBS solution; final concentration 0.5 mg/mL in 1 X PBS) was added. Cellular metabolism of MTT was permitted to occur at 370 in the presence of 5 % CO2. Following incubation, the MTT reagent was removed, 100 μL of dimethyl sulfoxide (DMSO) added and cells left to incubate for 10 mins in to dissolve formazan crystals. Samples were read at OD595nm using the SpectraMax190 Absorbance Microplate Reader and SoftMax® Pro Computer Software (Molecular Devices). Proliferation rates are expressed as a percentage of cell growth compared to 24 h.
5
Characterization of Organometallic Complexes
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MRI experiments were conducted at 3.0 T (GE Healthcare, SIGNA Architect, Chicago, IL, USA) and 9.4 T (BioSpec 94/20 USR; Bruker BioSpin, Ettlingen, Germany). Low resolution and high resolution with fast atom bombardment (FAB) ionization (JMS 700 Jeol; Akishima, Tokyo, Japan) was used for the characterization of Cu-DOTA-click-SF, Gd-azido-DOTA, and Gd-DOTA-click-SF formations. Matrix-assisted laser desorption/ionization coupled to time-of-flight mass spectrometry (MALDI-TOF MS) (MALDI-TOF/TOFTM 5800 system; AB Sciex) was used for the confirmation of the reaction between elastase and Gd-DOTA-click-SF. A SpectraMax® 190 absorbance microplate reader (Molecular Devices, San Jose, CA, USA) was used. Inductively coupled plasma coupled to mass spectrometry (ICP/MS; 7700; Agilent, Santa Clara, CA, USA) was used.
6
Chondrocyte Proliferation Assay with LNPs
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The cell proliferation assay was performed using BrdU enzyme linked immunosorbent assay (colorimetric) Kit (Roche Applied Science, Indianapolis, IN, USA) and assays were repeated at least three times. Briefly, chicken chondrocytes were cultured in 96-well plates at a density of 5,000 cells/100 μL/well in complete growth media. After 24 hours, LNPs (2.5 μM) were added to each well at different concentrations of 0% (blank), 1% (v/v), 2% (v/v), and 4% (v/v). The cells were labeled using 10 μL BrdU per well (10 μM BrdU) for 2 hours, before the cell proliferation assay was performed on days 1 and 2. After incubation with BrdU, the cell DNA was denatured in one step by adding FixDenat. Next, the cells were incubated with the anti-BrdU-POD antibody for 90 minutes at room temperature. After removal of the antibody conjugate, the cells were washed and the substrate solution was added. The reaction product was quantified by measuring the absorbance using a SpectraMax 190 Absorbance Microplate Reader (Molecular Devices, Sunnyvale, CA, USA) at 370 nm with a reference wavelength of 492 nm.
7
Cell Proliferation Quantification Protocol
8
Quantification of IL-1β secretion
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Cells were seeded in 12‐well plate at 105 cells per well and cultured for 48 h until confluency (#665180, Greiner Bio‐One, Monroe, NC, USA). Media were then aspirated and 500 μL of fresh media was added, and cells were incubated for another 24 h. Supernatant was used for human IL‐1β quantification using Quantikine ELISA kit (#DLB50, R&D Systems, Minneapolis, MN, USA), according to the manufacturer's protocol. Plate was read with SpectraMax 190 absorbance microplate reader (Molecular Devices, San Jose, CA, USA) using the softmax pro software v5.4.1 (Molecular Devices). IL‐1β quantification was measured five times per cell line.
9
Ultrasound-Treated Cell Viability Assay
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Cell viability was evaluated using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) kit (Beyotime, Wuhan, China) [26 (link)]. Cell suspension treated with ultrasound was grown in 96-well plates. Cells were incubated with 20 μL MTT reagent in each well for 4 h at 37°C, which was subsequently replaced by 100 μL DMSO. Following 10-min agitation on a plate shaker, the optical density of the samples was measured at a wavelength of 490 nm (A490) using a SpectraMax 190 Absorbance Microplate Reader (Molecular Devices, Sunnyvale, CA). The results were calculated using the following equation: Cell viability (%)=(A490 of experimental group/A490 of control group)×100%.
10
Plasma FGF21 Quantification in Mice
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Blood samples were collected with cardiac puncture after mice were sacrificed using carbon monoxide gas. Measurement of alanine transaminase (ALAT) was performed at the Laboratory of the Department of Equine and Small Animal Medicine, the Faculty of Veterinary Medicine, University of Helsinki. From approximately half of the blood volume, plasma was separated by centrifugation of blood at 3,000 g for 5 min at +4°C, transferred to 1.5-ml tubes, and maintained at −80°C until analysis. From serum, FGF21 (Quantikinew Mouse FGF-21, R&D Systems) was quantified by ELISA assay. Microplates were read by Spectra-Max 190 Absorbance Microplate Reader (Molecular Devices, Inc., USA) and analyzed by SoftMax Pro (Molecular Devices, Inc.).
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