Apoptosis was determined using a Cell Death Detection ELISA assay (Roche Diagnostics, Rotkreuz, Switzerland), based on the detection of cytoplasmic histone-associated DNA fragments in apoptotic cells. Cells were plated in 96-well plates in the appropriate medium (1 × 104 cells per well). After 24 h the cells were serum-deprived for 24 h followed by incubation with 100 µM hydrogen peroxide for 24 h. After fixation of cell lysates, the amount of cell death was quantified using the Cell Death Detection ELISA assay according to the manufacturer´s specifications. Quantification was performed by measuring the absorbance at 405 and 490 nm using a Power Wave 340 ELISA reader (Bio-TEK Instruments, Winooski, USA).
Power wave 340 elisa reader
Manufactured by Agilent Technologies
Sourced in United States
The Power Wave 340 ELISA reader is a versatile and reliable instrument designed for performing enzyme-linked immunosorbent assays (ELISA). It features a photometric detection system that can measure the absorbance of samples in microplates, allowing for accurate quantification of analytes in various applications.
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5 protocols using power wave 340 elisa reader
1
Quantification of Apoptosis by ELISA
2
BrdU-based Cellular Proliferation Assay
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DNA synthesis as a measure of cellular proliferation was obtained by a 5-bromodeoxyuridine (BrdU)-incorporation assay (Cell Proliferation ELISA, Roche Diagnostics, Rotkreuz, Switzerland). Cells were cultured in 96-well plates (1 × 104 cells per well) in the appropriate medium. After 24 h of serum-deprivation, cells were incubated with the indicated factors for 24 h. BrdU incorporation was carried out according to the manufacturer´s specifications. Quantification was performed by measuring the absorbance at 370 and 492 nm using a Power Wave 340 ELISA reader (Bio-TEK Instruments, Winooski, USA).
3
Monocyte CD45 Phosphatase Activity Assay
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CD14+ monocytes were incubated in RPMI containing GM-CSF and IL-4 with or without IL-10 for 30 min. The activity of CD45 PTPase was determined by a Human Active CD45 activity assay (R&D Systems) according to the manufacturer’s instructions. Briefly, 5×106 CD14+ monocytes in 200 μl lysis buffer and 100 μl lysate were applied to the assay plate. Absorbance at 620 nm was measured using the Biotek Powerwave 340 ELISA reader (BioTek Instruments, Inc., Winooski, VT, USA).
4
Ferric Reducing Antioxidant Power of C. barometz
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The ferric-reducing antioxidant power (FRAP) of the ethanol extract of C. barometz was assessed according to the method mentioned with minor modifications in Benzie et al’s study.22 (link) The FRAP reagent was prepared freshly from acetate buffer (pH 3.6), 10 mM TPTZ [2,4,6-Tri(2-pyridyl)-s-triazine] solution in 40 mM HCl and 20 mM Fe (III) chloride solution in the proportion of 10:1:1 (v/v), respectively. Butylated hydroxytoluene (BHT), ascorbic acid, quercetin, and gallic acid were used as controls; 10 µL of plant extract, standard, and controls were added to 300 µL of the FRAP reagent (triplicate) and left in the dark for 4 minutes. Then the absorbance was recorded at 593 nm using a spectrophotometer of power wave ×340 ELISA Reader (Bio-Tek Instruments, Inc., Winooski, VT, USA). The standard curve was created linearly (R2 =0.998) between 100 and 1,000 M FeSO4.
5
ABTS Radical Scavenging Activity Assay
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The ABTS radical scavenging activity was evaluated according to method described elsewhere [53 (link)]. The ABTS reagent was generated by mixing 5 mL of 7 mM ABTS solution with 88 μL of 140 mM potassium persulfate in the dark at room temperature for 16 h in order to complete the formation of radicals. The solution was then diluted with 95% ethanol such that the absorbance at 734 nm fell within the range 0.70 ± 0.05. To analyze the scavenging activity, 100 μL ABTS reagent was mixed with 100 μL of each sample solution. The mixture was reacted at room temperature for 6 min and the absorbance was read at 734 nm using a PowerWave 340 ELISA reader (Bio-Tek Instruments). The blank was prepared according to the same protocol using distilled water rather than the sample. Equation (4) was used to calculate the scavenging activity of ABTS radicals:
where Acontrol denotes the absorbance of ABTS without the sample and Asample represents the absorbance of ABTS with the tested samples. Trolox was prepared as a standard. A calibration curve showing the scavenging percentage against the various concentrations of Trolox standard was created. The ABTS radical scavenging activity of the samples was expressed as Trolox equivalent antioxidant capacity (TEAC), indicating the concentration (μM) of Trolox.
where Acontrol denotes the absorbance of ABTS without the sample and Asample represents the absorbance of ABTS with the tested samples. Trolox was prepared as a standard. A calibration curve showing the scavenging percentage against the various concentrations of Trolox standard was created. The ABTS radical scavenging activity of the samples was expressed as Trolox equivalent antioxidant capacity (TEAC), indicating the concentration (μM) of Trolox.
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