We measured the BDNF levels using a human BDNF ELISA kit (Life Technologies Corporation Carlsbad, CA, USA). They were detected using a Biochrom Anthos Zenyth 200 rt Microplate Reader (Cambridge, UK).
Anthos zenyth 200rt microplate reader
Manufactured by Harvard Bioscience
Sourced in United Kingdom
The Anthos Zenyth 200rt Microplate Reader is a compact and versatile instrument designed for absorbance measurements in a variety of microplate formats. It features a wavelength range of 340 to 750 nm and can accommodate 6- to 384-well plates.
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7 protocols using anthos zenyth 200rt microplate reader
1
Measuring BDNF Levels with ELISA
2
Yeast α-Glucosidase Inhibition Assay
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The enzyme inhibition activity against yeast α-glucosidase (Sigma-Aldrich, USA) were determined using 1 mM of p-nitrophenyl-α-D-glucopyranoside (PNPG) as substrate according to Wan et al.[12 (link)] with minor modifications. In 96 well plate, 30 μl of enzyme solution (0.5 U/ml), 30 μl of 0.1 M sodium phosphate buffer (pH 6.9) and 30 μl of tested inhibitors (mango, mangiferin [MIRA, China], or acarbose [Sigma-Aldrich, USA]) in dimethyl sulfoxide (DMSO) were mixed and incubated at 37°C for 10 min. Next, 30 μl of substrate were added and incubated again at 37°C for 20 min. After incubation, 80 μL of 0.2 μM Na2 CO3 was added to stop the reaction. The absorbance was measured at 405 nm using Anthos Zenyth 200 RT microplate reader (Biochrom, England). All tested inhibitors were analyzed in triplicate. The percent inhibition was calculated by the following formula:
3
Quantification of Soluble CD26 and CD66e
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Plasma samples were assayed for soluble CD26 and CD66e using separate ELISA kits for CD26 (ab222872, Abcam, USA) and CD66e (ab99992, Abcam, USA) in accordance with the instructions of manufacturers. Briefly, 50 μL of each sample and serially diluted standards provided were loaded into a 96-well CD26 or CD66e antibody precoated plates. This was followed by incubation for 2 hours (hrs) at room temperature. The plates were then washed 5 times with the buffer and 50 μL of biotinylated anti-human monoclonal antibody was added and incubated for 1 hr. The plates were washed again 5 times with the buffer and 50 μL of streptavidin-horseradish peroxidase conjugate was dispensed in each well and incubated at room temperature for 30 minutes (min). After washing, 50 μL of substrate solution (TMB) was added and finally the reaction was stopped using 50 μL of H2SO4 after 30 min. The colorimetric signal was measured by absorbance at 450 nm using the Anthos Zenyth 200rt microplate reader (biochrom). The limit of detection for CD26 is 50 ng/mL whereas the detection limits for CD66e is 0.2 ng/mL.
4
Evaluating Drug Effects on MSC Viability
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MSCs were seeded in a 24-well plate at 5000 cells/well and cultivated in the presence of drugs for 24 h or 7 d. After that, MSCs were incubated with standard medium containing 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT; Sigma-Aldrich, MO, USA) (final concentration of 555.56 μg/ml) for 4 h at 37°C. At the end of the experiment, the medium was removed, and DMSO was added. The absorbance was read at 570 nm in a 96-well plate using a microplate reader (Biochrom Anthos Zenyth 200rt microplate reader, Cambridge, UK). Experiments were performed in quadruplicate from four independent assays [48 (link),54 (link)].
5
Quantifying Soluble Amyloid-Beta Levels
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We measured soluble Aβ40 and Aβ42 levels in the hippocampus and post-hypothalamus using Ultrasensitive Rat Aβ40 and Aβ42 ELISA kits (Arigo Biolaboratories Corp, Hsinchu, Taiwan), in accordance with manufacturer instructions. Expression levels were detected using a Biochrom Anthos Zenyth 200rt Microplate Reader (Cambridge, UK).
6
Brainstem Cytokine Quantification
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The NTS of the brainstem were sectioned and homogenized with T-PER (Thermo Fisher, Waltham, MA, USA) containing protease and phosphatase inhibitors cocktail at 4 °C. The total protein was harvested by grinding and centrifugation. The total protein content was quantitatively analyzed by Coomassie R-250 (Thermo Fisher, Waltham, MA, USA). The concentration of IL-1β, IL-6, TNF-α, and fractalkine of serum, CSF, or NTS protein lysate were measured by ELISA kit, performed according to the manufacturer’s instruction (Cloud-Clone Corp, Katy, TX, USA). Expression values were detected by Anthos Zenyth 200rt Microplate Reader (Biochrom, Cambridge, UK). The final values were calculated and normalized to NTS protein mass.
7
Fructose and Glucose Levels in Rat Samples
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Heart blood and cerebrospinal fluid (CSF) samples were collected after the rats were sacrificed with excess CO2. Fasting fructose levels in serum and CSF (EFRU-100; EnzyChrom Fructose assay kit) as well as fasting glucose levels in CSF (EBGL-100; EnzyChrom glucose assay kit) were measured using an assay kit (BioAssay System, Hayward, CA, USA) and detected using a Biochrom Anthos Zenyth 200rt Microplate Reader (Cambridge, UK). Levels of serum total cholesterol, direct low-density lipoprotein (LDL), direct high-density lipoprotein (HDL), triglycerides, and glucose were determined using a Clinical Chemistry Analyzer (Ortho Clinical VITROS™ 350 System, Raritan, NJ, USA).
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