To determine AChE activity in the cortex and in the hippocampus, the ELISA kit ABCAM® ImmunoAssay System, AB138871 was used. The assay was performed according to the manufacturer’s instructions. AChE activity was measured at 410 nm using Gen 5 2.0 software and a Biotek Eon spectrophotometer.
Eon spectrophotometer
Manufactured by Agilent Technologies
Sourced in United States
The Eon spectrophotometer is a high-performance instrument designed for accurate and reliable absorbance measurements. It features a wide wavelength range, high-resolution optics, and user-friendly software for efficient data analysis.
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Lab products found in correlation
Enzymatic kit, by Sekisui (4 mentions)
Kinetic assay, by Sekisui (4 mentions)
Elisa, by Calbiotech (3 mentions)
Elisa kit against human bdnf, by Boster Bio (2 mentions)
Nitrocefin, by Merck Group (1 mentions)
Take3 micro volume plate, by Agilent Technologies (1 mentions)
Qiaamp dna kit, by Qiagen (1 mentions)
Quantasoft version 1, by Bio-Rad (1 mentions)
Qx200 autodg droplet digital pcr system, by Bio-Rad (1 mentions)
Elisa kit, by R&D Systems (1 mentions)
Insulin kit, by Merck Group (1 mentions)
Hotstar hifidelity dna polymerase, by Qiagen (1 mentions)
Hotstar hifidelity pcr buffer, by Qiagen (1 mentions)
Ez seq service, by Macrogen (1 mentions)
Qiaquick pcr purification kit, by Qiagen (1 mentions)
Creatine assay kit, by Abcam (1 mentions)
Simplestep elisa kit, by Abcam (1 mentions)
Whatman 541 filter paper, by Thermo Fisher Scientific (1 mentions)
Testosterone, by Calbiotech (1 mentions)
Ceramic beads, by Omni International (1 mentions)
33 protocols using eon spectrophotometer
1
Quantifying Cortical and Hippocampal AChE
2
Nitrocefin Hydrolysis Assay for Cells
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nitrocefin hydrolysis assays were performed as previously described with slight modifications [121 (link)]. Overnight cultures of cells harboring the pFBH1 plasmid containing an ampicillin resistance gene were subcultured 1:50 followed by regrowth to mid-log phase (~2 hours). Cells were washed (20 mM KPO4, pH 7.0, 1 mM MgCl2), resuspended to 109 CFU/mL, and combined with 100 μM nitrocefin (Sigma-Aldrich). Absorbance at 486 nm was recorded every minute for an hour using a BioTek Eon spectrophotometer.
3
BDNF Quantification by ELISA
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The amount of immobilized and released BDNF was quantified by an enzyme-linked immunosorbent assay (ELISA) kit against human BDNF (Boster Biological Technology Co., Ltd., Pleasanton, United States). The BDNF-ELISA kit was used in accordance to the manufacturer’s recommendations. A brief summary of the procedure follows. The 96-well plate delivered had been pre-coated with a monoclonal antibody for BDNF. Standards and samples were diluted in sample dilution buffer and added to the wells for 90 min. After the incubation, the plate content was discarded and a working solution of a biotinylated anti-human BDNF antibody was added for 60 min. After three washing steps with PBS, an incubation with a working solution containing an avidin-biotin-peroxidase complex followed (30 min). Further washing steps with PBS to remove the unbound complex were performed. For colorimetric detection 3,3′,5,5′-tetramethylbenzidine was added. The color development was stopped with 2 M sulfuric acid. All incubation steps were performed at 37°C. Absorbance was recorded at 450 nm on an EON spectrophotometer (Biotek, Winooski, United States).
4
ddPCR Determination of AMY1A Copy Number
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Copy number state of AMY1A gene from individuals in the MDCS study was previously determined with droplet digital polymerase chain reaction (ddPCR) using a QX200 AutoDG Droplet Digital PCR system (Bio-Rad laboratories), described in Rukh et al. [9 (link)]. Quality control of the measurements was done by repeated runs for randomly selecting samples (~ 10%) as well as samples with high copy numbers. Determination of copy number state of AMY1A in individuals included in cohort 2 was performed on DNA extracted from the brain tissue. Briefly, the DNA was purified using QIAamp® DNA kit (Qiagen) according to the manufacturer’s instruction. The DNA concentration and purity were measured using Take 3 Micro-Volume plate and Eon spectrophotometer (Biotek, Winooski, VT, USA) and the DNA quality was evaluated with 1% agarose gel electrophoresis. The genotyping was performed by ddPCR at TATAA Biocenter (Gothenburg, Sweden) on a QX200 AutoDG Droplet Digital PCR system (Bio-Rad Laboratories) with AP3B1 as the reference (2 copies). A negative control (no template) was included on each plate. Quality control and determination of copy number state was performed in Quantasoft version 1.7.4 (Bio-Rad Laboratories).
5
Evaluation of Inflammatory Biomarkers
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After the rats were anesthetized, blood was taken from the ophthalmic vein plexus and left to stand at 4°C. Then, the supernatant was collected by centrifugation. IL-6, IL-1β, TF, MCP-1, MPO, SOD, vWF, P-selectin, VCAM-1, ICAM-1 level in rat peripheral blood and HPAECs culture supernatants were determined as per the instructions of the ELISA kits (R&D Systems, Minneapolis, MN, USA). Optical Density (OD) at 450 nm was recorded using an Eon spectrophotometer (BioTek Instruments, Winooski, VT, USA). Experiments were performed independently in triplicate. Colorimetric activity assays were performed to determine plasma TF (Abcam), MPO (Sigma-Aldrich), SOD (Sigma-Aldrich), and vWF activity (Abcam) in rats at weeks 1, 2 and 4, respectively, according to the manufacturer's instructions.
6
BDNF Quantification via ELISA Protocol
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For the quantification of the amount of immobilized and released BDNF an enzyme-linked immunosorbent assay (ELISA) kit against human BDNF (Boster Biological Technology Co., Ltd., Pleasanton, USA) was applied. The BDNF-ELISA kit was used in accordance to the manufacturer´s recommendations. In brief, a monoclonal antibody for BDNF had been precoated onto the 96-well plate. Standards and samples were diluted in sample dilution buffer and were added to the wells. After an incubation period of 90 min, the plate content was discarded and a working solution of a biotinylated anti-human BDNF antibody was added for 60 min. This was followed by three washing steps with PBS and by the incubation with a working solution containing an avidin-biotin-peroxidase complex. Then, the unbound conjugates were washed off with PBS. Finally, the detection was performed with 3,3´,5,5´-tetramethylbenzidine and stopped with 2 M sulphuric acid. All incubation steps were performed at 37°C. Absorbance was read at 450 nm on an EON spectrophotometer (Biotek, Winooski, USA).
7
Serum Metabolite Evaluation
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Glycemia, lactate, triglycerides and total cholesterol levels were evaluated in serum samples using a colorimetric test (Labtest kit, Lagoa Santa, MG, Brazil, Ref. 133, 138, 87 and 76, respectively). Insulin was quantified using an immunoassay technique (enzyme-linked immunosorbent assay). The insulin kit was obtained from Millipore Corp. (Bedford, MA, USA, Ref. EZRMI-13 K). The tests were performed on an EON spectrophotometer (BioTek Instruments, Inc., Winooski, VT, USA) with the ideal optical density established in the manufacturing manual of each kit.
8
Plasma Hormones and HMB Analysis
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Plasma HMB was analyzed by Metabolic Technologies Inc. by gas chromatography-mass spectrometry with previously outlined methods to confirm HMB appearance in the plasma [22 (link)].
Plasma testosterone (cat. number: KGE010), serum growth hormone (cat. number: DGH00), and plasma IGF-1 (cat. number: DG100) were assayed via commercial kits (R&D Systems Minneapolis, MN, USA). Serum insulin was also assayed via a commercial kit (RayBiotech, Inc., Norcross, GA, USA). Determination of serum immunoreactivity values was determined using a BioTek Eon spectrophotometer (BioTek, Winooski, VT, USA). To eliminate interassay variance, all samples for a particular assay were thawed once and analyzed in the same assay run by a single technician and intra-assay variance for the hormones was 5.8%, 5.3%, 4.1%, and 4.3% for testosterone, growth hormone, insulin, and IGF-1, respectively.
Plasma testosterone (cat. number: KGE010), serum growth hormone (cat. number: DGH00), and plasma IGF-1 (cat. number: DG100) were assayed via commercial kits (R&D Systems Minneapolis, MN, USA). Serum insulin was also assayed via a commercial kit (RayBiotech, Inc., Norcross, GA, USA). Determination of serum immunoreactivity values was determined using a BioTek Eon spectrophotometer (BioTek, Winooski, VT, USA). To eliminate interassay variance, all samples for a particular assay were thawed once and analyzed in the same assay run by a single technician and intra-assay variance for the hormones was 5.8%, 5.3%, 4.1%, and 4.3% for testosterone, growth hormone, insulin, and IGF-1, respectively.
9
Biomarker Quantification in Serum Samples
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Creatine Kinase (CK) was analyzed with the use of a spectrophotometer and a commercially available enzymatic kit (Sekisui Diagnostics, Charlottetown, PE, Canada) per manufacturer's instructions. Myoglobin concentrations were determined using enzyme-linked immunosorbent assays (ELISA) (Calbiotech, Spring Valley, CA). Determination of serum immunoreactivity values was determined using a BioTek Eon spectrophotometer (BioTek, Winooski, VT, USA). All samples were run in duplicate with a mean intra-assay variance of 2.6% for CK and 5.73% for myoglobin. Coefficient of variation for each hemoglobin assay was 3.73% for and 0.65% for hematocrit.
Serum samples were assayed for concentrations of TNF-α, using a cytokine assay (Millipore Milliplex, cat no. HCYTOMAG-60K; Billerica, MA) on a MAGPIX instrument (Luminex Technologies; Luminex, Austin, TX) according to the manufacturer's instructions. All samples were run in duplicate with a mean intra-assay variance of 8.36% for TNF-α.
Serum samples were assayed for concentrations of TNF-α, using a cytokine assay (Millipore Milliplex, cat no. HCYTOMAG-60K; Billerica, MA) on a MAGPIX instrument (Luminex Technologies; Luminex, Austin, TX) according to the manufacturer's instructions. All samples were run in duplicate with a mean intra-assay variance of 8.36% for TNF-α.
10
Quantifying Phycocyanin Concentration and Purity
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Phycocyanin concentration and purity were determined by spectrophotometry as described by Bennet and Bogard 1973 [44 (link)]. Concentration was calculated with the formula below using absorbance at 620 nm and 652 nm with a Biotek Eon™ spectrophotometer with a high-performance microplate. For purity, a ratio of A620/A280 was used.
Phycocyanin absorbs light at about 620 nm and emits fluorescence at about 640 nm [45 (link)]. The purity of C-PC is usually evaluated using an absorbance ratio of A620/A280, and a purity of 0.7 is considered as food grade [44 (link)], Absorbance at 620 nm indicates maximum C-PC absorption, while at 280 nm, it is due to the presence of other proteins in the solution [46 (link)]. C-PC purity is a ratio regarding the contamination of other proteins if present in the same sample.
Phycocyanin absorbs light at about 620 nm and emits fluorescence at about 640 nm [45 (link)]. The purity of C-PC is usually evaluated using an absorbance ratio of A620/A280, and a purity of 0.7 is considered as food grade [44 (link)], Absorbance at 620 nm indicates maximum C-PC absorption, while at 280 nm, it is due to the presence of other proteins in the solution [46 (link)]. C-PC purity is a ratio regarding the contamination of other proteins if present in the same sample.
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