To measure the catalytic activity of the probe, a 10 µL Ab (HPR) solution (100 μg/mL) was added to 220 µL of 2% BSA solution, and a 200 µL AuNPs solution was added to 220 µL of 2% BSA solution. Then, the two mixtures were centrifuged at 15,000 rpm for 20 min at 4°C. The precipitate was washed with a BR buffer (containing 1% BSA and 0.05% Tween 20) and resuspended in 220 µL of 1% BSA, respectively. Then, 100 µL of the freshly prepared Ab (HRP) solution, AuNPs solution, and Ab (HRP)-AuNPs probe were collected (2.3 section) into three micro-wells of 96-well plates and incubated at 37°C for 1 hour, followed by washing three times. Then, the TMB enzyme substrate (100 μL) was added to each well. After 15 minutes of incubation at room temperature, the enzymatic reaction was stopped with 50 μL of a stop solution (2 M H2SO4), and the reaction product was quantified at A450 (OD450) by using an automatic plate reader (Bio-Tek Instruments, Inc., Winooski, VT, USA). The data were measured in triplicate, and the mean OD450 value and standard deviation for each sample were calculated.
Automatic plate reader
Manufactured by Agilent Technologies
Sourced in United States
The Automatic Plate Reader is a lab equipment designed to automatically read and analyze the contents of microtiter plates. It is capable of measuring absorbance, fluorescence, and luminescence signals from samples in the wells of the plate. The device can be used in a variety of applications, such as drug discovery, enzyme activity assays, and cell-based assays.
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8 protocols using automatic plate reader
1
Catalytic Activity Assay of Ab-AuNPs Probe
2
NF-κB Reporter Assay Protocol
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A luciferase reporter assay was performed using a Dual Luciferase Reporter Assay Kit (BPS Bioscience, San Diego, CA, USA), according to the manufacturer’s protocol. The luciferase reporter construct was co-transfected with NF-κB reporter into HaCaT cells using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA), according to the manufacturer’s guidelines. TCDD was administered after 24 h. To obtain the normalized luciferase activity for the NF-κB reporter, we subtracted the background luminescence and calculated the ratio of firefly luminescence from the NF-κB reporter to Renilla luminescence from the control Renilla luciferase vector. The relative luciferase activity was measured using an automatic plate reader (BioTek Instruments, Seoul, Korea).
3
Serum and Bone Antioxidant Analysis
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Venus blood was collected and transferred to a centrifuge tube. Serum was prepared by centrifuging at 3000g for 10 minutes and stored at −80°C. The right tibia was washed to allow for bone marrow collection. Serum malondialdehyde (MDA), serum superoxide dismutase (SOD), and bone glutathione reductase (GSR) were measured using commercial kits and all procedures were performed according to the manufacturers' instruction (Nanjing Jiancheng Biological Bioengineering Co., Nanjing, China). An automatic plate reader (BioTek Instruments Inc., Winooski, VT, USA) was introduced to the measurements. Part of the bone marrow was centrifuged to obtain bone marrow cells. These cells were then labelled with 2′,7'-dichlorfluorescein-diacetate (20M) for 30 minutes at 37°C and followed by measurement of fluorescence intensity using BD FACSCalibur flow cytometry (BD Biosciences, San Jose, CA, USA) [39] (link). Data were analysed and acquired using CellQuest Pro software (BD Biosciences).
4
ELISA Detection of PCV2 Antibodies
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In this method, 100 μL of the anti-PCV2 capsid antibodies (10 μg/mL) in a coating buffer was immobilized in each well and incubated at 4°C overnight. The solutions were discarded and washed three times to remove the unbound antibodies. Then, 100 μL of the blocking buffer (5% BSA) was injected into the wells and kept at 37°C for 2 hours, thereby blocking the nonspecific binding sites. The wells were washed again, and 100 μL of the PCV2 in the PBS was added to the wells. The plate was kept at 37°C for 1 hour and washed three times. Later, 100 μL of the Ab (HRP)-AuNPs probe was added to each well and incubated at 37°C for 1 hour, followed by three times washing. Finally, TMB enzyme substrate (100 μL) was added to each well. After 15 minutes of incubation at room temperature, the enzymatic reaction was stopped with 50 μL of a stop solution (2 M H2SO4), and the reaction product was quantified at A450 (OD450) by using an automatic plate reader (Bio-Tek Instruments, Inc., Winooski, VT, USA). The data were measured in triplicate, and the mean OD450 value and standard deviation for each sample were calculated.
5
Cytotoxic Effect of APA on Tumor Cells
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The cytotoxic effect of APA on tumor cells was determined using 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazoliumbromide (MTT) staining (28). Briefly, 5 × 103 cells/well were plated in 96-well plates with 100 μL of culture medium for 24 h and then exposed to different concentrations of APA. After 72 h, the culture medium was replaced with 100 μL of fresh medium including 0.5 mg/mL MTT. Following 4 h of incubation 37°C, this medium was removed, and 100 μL of DMSO was added to each well to dissolve the purple formazan crystals. The color reaction was quantified using an automatic plate reader (Bio-Tek Instrument Inc, Winooski, VT) at 570 nm. The half maximal inhibitory concentration (IC50) (in μM), which represents the concentration of the drug that lowers the cell number by 50%, was calculated from the concentration-response curve. Each experiment was repeated three times (n = 3), and the average IC50 shown.
6
TCDD and pAhRR Cytokine ELISA
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IL-6, IL-22, and S100A7 quantitation by enzyme-linked immunosorbent assay (ELISA) protein expression levels in HPDFs after treatment with TCDD and pAhRR were evaluated by ELISA. HaCaT cells were exposed to various concentrations of TCDD and pAhRR for 24 h, and the supernatant from the cultured cells was collected. IL-6, IL-22, and S100A7 levels were measured using ELISA (Biolegend, San Diego, CA, USA). Briefly, each well was blocked with blocking buffer for 2 h and washed with buffer. Antibodies against IL-6, IL-22, and S100A7 were added to the media and incubated for 1 h. A substrate solution and stop solution were introduced sequentially, and the optical density of each well was determined within 30 min using an automatic plate reader (BioTek Instruments Korea Ltd.).
7
ELISA for Porcine IgG Detection
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96-well microtiter plates manufactured from polyvinyl chloride were coated with 50 μl GP5 protein in carbonate buffered saline (CBS, pH 9.6) and incubated overnight at 4 o C. After three washes in PBS containing 0.05% Tween 20 (PBST), the plates were incubated with blocking buffer (5% skimmed milk) for 1 h at 37 o C and with 50 μl serum samples diluted in PBS containing 5% E. coli lysate for 30 min at 37 o C, with each sample in duplicate. After washing six times with PBST, the plates were incubated with 50 μl HRP-conjugated goat anti pig IgG for 30 min at 37 o C. Then, the plates were washed again and finally incubated with 3,3',5,5'-tetramethylbenzidine (TMB, Sigma) for color development. After 10 min of incubation at room temperature, the enzymatic reaction was stopped with 2 M H 2 SO 4 and the horseradish peroxidase product was quantified at A450 (OD 450 ) with an automatic plate reader (Bio-Tek Instruments, Inc., Winooski, VT, USA). All data were measured in triplicate, and the mean value of OD 450 values and standard deviation for each sample were calculated.
8
Antioxidant Capacity Determination by ORAC
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Antioxidant activity was determined by the method of oxygen radical absorbance capacity (ORAC) by fluorescence detection (λ exc 485 nm and λ em 520 nm) using an automatic plate reader (BioTek Instruments), previously described (Cáceres et al., 2014b) . Sample replicates were independently analyzed and results were expressed as mg of Trolox equivalents (TE)/100g of sample d.w.
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