The MTT assay was used for three experiments. First, the cell cytotoxicity of HEPS was measured by plating exponentially growing PC12 cells at a density of 5 × 104 cells/well in 96-well plates, which were exposed with or without 25, 50, 100, 200, 250 μg/mL of HEPS for 24 and 48 h. The second stage of the assay measured cell cytotoxicity of Aβ1-40 (Sigma-Aldrich, USA) by adding 1.2 μM Aβ1-40 to PC12 cells for 24 and 48 h. The third stage was a cell protection assay, in which PC12 cells were incubated with 25, 50, 100, 200, 250 μg/mL of HEPS for 24 h, and 1.2 μM Aβ1-40 was added for 24 and 48 h. After each of these three experiments, the cells were incubated with 2 mg/mL 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) for 4 h at 37 °C, the media was carefully removed and 100 μl of DMSO was added to each well. Dark blue formazan crystals formed, the intact cells were solubilized for 30 min, and the absorbance at 570 nm was measured with a PowerWave XS ELISA reader (Bio-Tek, USA). The results were expressed as the percentage of MTT reduction, assuming the absorbance of control cells was 100 %.
Aβ1 40
Manufactured by Merck Group
Sourced in United States, China
Aβ1-40 is a laboratory peptide product. It is a synthetic fragment of the amyloid-beta protein consisting of the first 40 amino acids. This product is commonly used in research applications.
Automatically generated - may contain errors
Lab products found in correlation
Aβ1 42, by Merck Group (5 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Aβ25 35, by Merck Group (2 mentions)
Power wave xs elisa reader, by Agilent Technologies (1 mentions)
Inverted phase contrast microscope, by Olympus (1 mentions)
Streptomycin, by Thermo Fisher Scientific (1 mentions)
Penicillin, by Thermo Fisher Scientific (1 mentions)
96 well plate, by Corning (1 mentions)
Dmem f12 medium, by Thermo Fisher Scientific (1 mentions)
Arpe 19 hpv 16, by American Type Culture Collection (1 mentions)
Thioflavin t, by Merck Group (1 mentions)
Aβ1 40 peptide, by Merck Group (1 mentions)
Human insulin, by Merck Group (1 mentions)
P akt, by Santa Cruz Biotechnology (1 mentions)
Dmem f12 cell culture medium, by Thermo Fisher Scientific (1 mentions)
Trypsin edta, by Thermo Fisher Scientific (1 mentions)
2 7 dichlorofluorescein diacetate dcfh da, by Merck Group (1 mentions)
Bcl2 associated x (bax), by Santa Cruz Biotechnology (1 mentions)
Bcl 2, by Santa Cruz Biotechnology (1 mentions)
Aβ1 42, by Fujirebio (1 mentions)
25 protocols using aβ1 40
1
Evaluation of HEPS Neuroprotection Against Aβ
2
RPE Cell Line Aβ1-40 Exposure Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The human RPE cell line (ARPE-19/HPV-16) was purchased from the American Type Culture Collection. The cell line was cultured at 37°C with 5% CO2 in an incubator (Thermo Fisher Scientific, Inc.) with DMEM/F-12 medium (cat. no. 11330057; Gibco; Thermo Fisher Scientific, Inc.) containing 10% fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc.) and 1% 10,000 U/ml penicillin/10,000 µg/ml streptomycin (Gibco; Thermo Fisher Scientific, Inc,.). The cells were subcultured every 3 days in a 35-mm culture flask (Corning Inc.). After the cells (2×104 cells/well) were seeded in 96-well plates (Corning Inc.), and different concentrations of Aβ1-40 (0, 0.01, 0.1, 0.5, 1, 5 and 10 µmol/ml; Sigma-Aldrich; Merck KGaA) mixed with culture medium were added to the cells in order to establish OS, the cells were incubated at 37°C for 24 h. For subsequent experiments, 0.5 µmol/ml Aβ1-40 was used. Following culture for 24 h, the morphology of cells in the control and 0.5 µmol/ml Aβ1-40 groups was observed under an inverted phase contrast microscope (magnification, ×200; Olympus Corporation).
3
Amyloid Aggregation Assay Protocols
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Ubiquitin from bovine erythrocytes, recombinant Human Insulin and Thioflavin T were purchased from Sigma Aldrich and used without further purification. Aβ(1-40) was purchased from Biopeptide and pre-treated as previously reported 64 .
All experiments on UBQ samples were performed in 20 mM sodium phosphate buffer, pH 6.5. The sample was freshly prepared and filtered through 0.20 μm filters (17761, Sartorious), just before the measurements. Different protein concentrations had been used: 0.5 mg/ml, 0.7 mg/ml and 1.8 mg/ml. Protein concentration was spectrophotometrically determined using a molar extinction coefficient of 1254 dm 3 mol -1 cm -1 at 280 nm 31 . Human Insulin powder (Sigma-Aldrich) and Aβ (1-40) peptide (prepared as described in 65 ), were dissolved in 20mM sodium phosphate buffer, pH 6.5.
Protein concentration of Human Insulin and Aβ(1-40) peptide were estimated by means of absorbance measurements using a molar extinction of 1.067 cm -1 (mg/ml) -1 at 276 nm 66 , and of 1390 M -1 cm -1 at 276nm, respectively 67 .
In order to obtain aggregates, 0.2 mg/ml Human Insulin in 20 mM sodium phosphate buffer, pH 6.5, was incubated at 60°C stirring at500 rpm for 24 hours, and 0.2 µM Aβ(1-40) in 20 mM sodium phosphate buffer, pH 6.5, was incubated at 37 degrees with stirring 500 rpm for 24 hours.
Resulting aggregates were found to be positive to Thioflavin T test.
All experiments on UBQ samples were performed in 20 mM sodium phosphate buffer, pH 6.5. The sample was freshly prepared and filtered through 0.20 μm filters (17761, Sartorious), just before the measurements. Different protein concentrations had been used: 0.5 mg/ml, 0.7 mg/ml and 1.8 mg/ml. Protein concentration was spectrophotometrically determined using a molar extinction coefficient of 1254 dm 3 mol -1 cm -1 at 280 nm 31 . Human Insulin powder (Sigma-Aldrich) and Aβ (1-40) peptide (prepared as described in 65 ), were dissolved in 20mM sodium phosphate buffer, pH 6.5.
Protein concentration of Human Insulin and Aβ(1-40) peptide were estimated by means of absorbance measurements using a molar extinction of 1.067 cm -1 (mg/ml) -1 at 276 nm 66 , and of 1390 M -1 cm -1 at 276nm, respectively 67 .
In order to obtain aggregates, 0.2 mg/ml Human Insulin in 20 mM sodium phosphate buffer, pH 6.5, was incubated at 60°C stirring at500 rpm for 24 hours, and 0.2 µM Aβ(1-40) in 20 mM sodium phosphate buffer, pH 6.5, was incubated at 37 degrees with stirring 500 rpm for 24 hours.
Resulting aggregates were found to be positive to Thioflavin T test.
4
Aβ1-40 Quantification in Brain Homogenates
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total brain homogenates were used for all the ELISA. For the Aβ1–40 (Millipore cat#EZHS40) we analyzed 50 μg per well following the protocol provided.
5
Alzheimer's Disease In Vitro Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Aluminium chloride hexahydrate, Maltol, Tox 7 kit, (3-(4, 5-dimethyl- thiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) dye and 2,7-dichlorofluorescein diacetate (DCFH-DA) were purchased from Sigma Chemical Co. Ltd. (St. Louis, MO, USA). DMEM/F12 cell culture medium, trypsin–EDTA, fetal bovine serum (FBS) and 100X antibiotic and antimycotic solution were purchased from (Gibco, USA). Caspase 12 and p53 antibodies were purchased from Abcam (Cambridge, UK), while Nrf2, NQO1, pAKT, p21, Bax and Bcl2 were procured from Santa Cruz (Texas, USA). CHOP/GADD153 and ubiquitin antibodies were purchased from BioLegend (USA) and Aβ1-40 was purchased from Millipore (MA, USA). Caspase 9, 3 and 12 activity kits as well as caspase 3, 9 and 12 inhibitors were purchased from BioVision Inc. (Mountain View, CA) while pan-caspase inhibitor (QVD-OPh) was procured from Abcam. All other chemicals were obtained from Merck unless otherwise mentioned.
6
Drift Assessment in Amyloid-Beta Assays
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Finally, drift was assessed by analyzing QC samples at either end of the plate at five occasions (Innogenetics Aβ1-42) or by running the standard curve at both ends of the plate (Merck Millipore Aβ1-40).
7
Validating ELISA Assays for Amyloid-β
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
We performed a validation to evaluate the performance of the ELISA assays (i.e., Innogenetics Aβ1-42 and Merck Millipore Aβ1-40) with emphasis on within- and between plate imprecision, hook effects, cross reactivity, and drift across the plates. This validation was performed using QC samples at two concentrations (20 and 250 pg/mL for Aβ42, 25 and 250 pg/mL for Aβ40), prepared in sample buffer provided in the ELISA kit.
8
Antibody and ELISA Reagent Procurement
9
Precision Evaluation of Aβ ELISA
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Within plate precision was calculated from the analyses of six replicates of each QC-sample (in duplicate) at one occasion. Between plate precision was calculated from the same QC samples at six (Innogenetics Aβ1-42) or four (Merck Millipore Aβ1-40) different ELISA plates, at different days, using two different ELISA lots.
10
Alzheimer's Disease Neuroprotection Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
SL was purchased from Shanghai Taiwei Pharmaceutical Company Limited (China). Acetone insoluble substance was 93.9%. The main chemical compositions included 84.2% phosphatidylcholine, 2.4% phospholipid acyl amine, and no phosphatidylinositol. SIF was purchased from ZHT Sci-Tech (Beijing) Co., Ltd. (China). The total isoflavone content of SIF was 92.15%. The main chemical compositions included 17.64% genistein, 53.24% daidzein, and 21.27% glycitein. SL and SIF were dissolved in 0.5% CMC-Na. Synthetic Aβ1-40 and Aβ25-35 were purchased from Sigma-Aldrich (St. Louis, MO, USA). They were dissolved in N.S. and incubated at 37°C for three days to form aggregation [13 (link)].
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!