ECs were seeded at 3 × 105 cells/well in 12-well plates and cultured until confluent for 2 to 3 days. On the day of the assay, ECs were primed for 2 h with 100 μg/mL HDL before stimulating with 0.1 μM of Aβ40 and Aβ42 monomers at 37 °C for total association, or at 4 °C for cell surface binding. After 3 h, hCMEC/D3 were washed 3 times with PBC and lysed in RIPA buffer containing 10 mM Tris pH 7.4, 150 mM NaCl, 1.0% NP-40, 1.0````% sodium deoxycholate, 0.1% SDS and cOmplete protease inhibitor with EDTA (Roche). Aβ40 (KHB3442, Life Tech) and Aβ42 (KHB3482, Life Tech) were quantified using commercial ELISAs and normalized to total protein concentration. For Aβ uptake, hCMEC/D3 were seeded at 1 × 105 cells/well in 24-well plates and cultured to confluence for 2 to 3 days. On the day of the assay endothelial, ECs were primed for 2 h with 1 mg/mL of HDL before stimulating with 1 μM monomeric FITC-Aβ40 and FITC-Aβ42 (Bachem) prepared as described above. After 3 h at 37 °C, hCMEC/D3 were washed 3 times with PBS and fixed in 4% PFA for 20 min. After one Tris-HCl and two PBS washes, hCMEC/D3 were mounted in Prolong antifade reagent.
Khb3442
Manufactured by Thermo Fisher Scientific
Sourced in United States
The KHB3442 is a laboratory equipment product from Thermo Fisher Scientific. It is a device designed for performing specific analytical tasks in a laboratory setting. The core function of the KHB3442 is to provide precise and accurate measurements for research and testing purposes.
Automatically generated - may contain errors
Lab products found in correlation
Khb3482, by Thermo Fisher Scientific (15 mentions)
Khb3481, by Thermo Fisher Scientific (3 mentions)
Ethylene diamine tetraacetic acid (edta), by Roche (2 mentions)
Bullet blender storm 24, by Next Advance (2 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Khb7031, by Thermo Fisher Scientific (2 mentions)
Fitc aβ42, by Bachem (1 mentions)
Spectramax plus 384, by Molecular Devices (1 mentions)
Protease inhibitor cocktail, by Merck Group (1 mentions)
Infinite m200, by Tecan (1 mentions)
Formic acid, by Merck Group (1 mentions)
Bicinchoninic acid protein assay, by Thermo Fisher Scientific (1 mentions)
Guanidine hydrochloride, by Merck Group (1 mentions)
Aβ42 elisa kit, by Thermo Fisher Scientific (1 mentions)
Synergy h1 hybrid multi mode reader, by Agilent Technologies (1 mentions)
Complete protease inhibitor cocktail, by Roche (1 mentions)
Amicon ultra centrifugal filter, by Merck Group (1 mentions)
Ab100688, by Abcam (1 mentions)
Ab100750, by Abcam (1 mentions)
Ns830, by Merck Group (1 mentions)
31 protocols using khb3442
1
Quantification of Aβ Binding and Uptake in Brain Endothelial Cells
2
Quantification of Aβ Peptides
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Luminal medium was collected from the circulation chamber and 5 mm tissue rings were crushed and lysed in RIPA buffer (10 mM Tris pH 7.4, 150 mM NaCl, 1.0% NP-40, 1.0% sodium deoxycholate, 0.1% SDS and cOmplete protease inhibitor with EDTA (Roche, Switzerland)). Aβ40 (KHB3442, Life Tech, ThermoFischer Scientific) and Aβ42 (KHB3482, Life Tech) were quantified using commercial ELISAs and normalized to total protein concentration, measured by BCA.
3
Quantifying Soluble and Insoluble Amyloid-Beta 42
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Commercially available Aβ42 sandwich ELISA kits (Invitrogen, KHB3442) were used for analyzing the soluble and insoluble Aβ42 levels. The soluble fraction samples were diluted in the ratio of 1:200 and the insoluble fraction samples were firstly neutralized using neutralization buffer in the ratio of 1:20 and diluted further to in the ratio of 1:500 in the ELISA diluent buffer. The experiment was performed as the manufacturer’s protocol. Synthetic Aβ42 peptide in the assay kit was used for standard curve (y = 67.626x2 + 240.72x + 4.8938).
4
Aβ42 Protein Release in Macrophages
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At day 7, macrophages were incubated for 30 min or 24 h with fibrillar (f) or oligomeric (o) Aβ42 and thereafter culture medium was collected. Total protein levels in the supernatant were determined using the Pierce BCA Protein Assay Kit (#23227; Thermo Scientific). The Aβ42 levels in the supernatant were assessed using a sandwich ELISA for an anti-human Aβ42 end-specific kit (KHB3442, Invitrogen; which does not recognize mouse Aβ nor human Aβ40/Aβ43). The kit was used according to the manufacturer's instructions. This assay is based on a combination of two antibodies specific for the N- and the COOH-termini of Aβ42 sequences. The bound rabbit anti-COOH-terminus was detected with a horseradish peroxidase-labeled anti-rabbit antibody and was read at 450 nm using a microplate reader (Spectra Max 384 plus, Molecular Devices). Concentrations detected at 24 h incubation were normalized to basal levels at 30 min incubation.
5
Quantifying Aβ40 and Aβ42 in Plasma and Brain
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Aβ40 and Aβ42 levels in plasma and brain samples were determined by ELISA (KHB3482; KHB3442) according to the manufacturer’s protocol (Invitrogen, Carlsbad, CA, USA). Aβ40 and Aβ42 were extracted from brain tissue by homogenizing the samples with Tris–HCl buffer containing 5 M guanidine-HCl. Samples were diluted 1:20 in DPBS buffer containing 5% BSA and 0.03% Tween-20 and centrifuged at 16,000 g for 20 min at 4 °C. The supernatant was collected and diluted 1:1 before adding it to ELISA plates.
6
Quantification of Hippocampal Aβ, MDA, and SOD
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Hippocampal samples were homogenized in PBS (pH 8.0) and centrifuged at 3,000 g for 1 h at 4°C to remove insoluble material. The supernatant fractions were placed in ELISA plates for measurement of Aβ40 (KHB3482; Invitrogen, Carlsbad, CA, USA) and Aβ42 (KHB3442; Invitrogen).
Hippocampal samples were homogenized in PBS (pH 8.0) and centrifuged at 1,600 g for 10 min at 4°C to remove insoluble material. The supernatant fractions were placed in ELISA plates for measurement of methane dicarboxylic aldehyde (MDA) content (Beyotime, SO131) and SOD and Mn-SOD activities (Beyotime, SO103).
Hippocampal samples were homogenized in PBS (pH 8.0) and centrifuged at 1,600 g for 10 min at 4°C to remove insoluble material. The supernatant fractions were placed in ELISA plates for measurement of methane dicarboxylic aldehyde (MDA) content (Beyotime, SO131) and SOD and Mn-SOD activities (Beyotime, SO103).
7
Quantitation of Amyloid-β Peptides
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8
Brain Amyloid-Beta Extraction and Measurement
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Two-step sequential extraction of the brain Aβ using 2 % SDS and 70 % formic acid (FA; Sigma) was processed as described previously [20 (link)]. Briefly, cortical homogenate was mixed with equal volume of 4 % SDS in H-buffer containing protease inhibitor. The sample was then sonicated and centrifuged at 100,000 × g for 60 min at 4 °C. The supernatant was considered SDS-soluble fraction. The SDS-insoluble pellet was further resuspended in 70 % FA and centrifuged at 100,000 × g for 60 min at 4 °C. The supernatant was collected and neutralized with 1 M Tris, pH 11. SDS-soluble and SDS-insoluble fractions were stored at −80 °C until sandwich ELISA analysis. Aβ level was measured by a sensitive fluorescence based sandwich ELISA assay using a kit (Human β- Amyloid 1–42, Invitrogen KHB3442). The detailed experiments were performed according to the manufacturer’s protocol.
9
Enzyme-linked Immunoassays for Alzheimer's Biomarkers
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Specific p-Tau ser396 (Invitrogen, KHB7031) and Aβ1–42 (Invitrogen, KHB3442,) ELISA were performed according to manufacturer’s protocol as described [15 (link), 19 (link)]. Caspase-3 activity assays were performed according to manufacturer’s protocol as we previously described [14 (link), 18 (link)].
10
Quantification of Hippocampal Amyloid-Beta
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Whole hippocampi were snap frozen on dry ice and stored at -70°C until use. Frozen tissues were sonicated in lysis buffer (0.05 M Tris-HCl pH 7.5, 0.15 M NaCl, 1% NP-40, 1 mM Na-orthovanadate, 0.001% sodium fluoride, 1% protease inhibitor cocktail (Sigma)) and centrifuged to clear. The supernatants were transferred to new tubes and stored at -70°C. A solution of 8.2 M guanidine / 82 mM Tris HCl (pH 8.0) was added to the extracts to yield a solution with 5 M final guanidine concentration. Samples were diluted with 10x volume of PBS and centrifuged at 16,000 x g for 20 minutes at 4°C. The supernatant was carefully collected and stored on ice until analyses with the Aβ40 or Aβ42 ELISA kit from Invitrogen. ELISAs were performed according to manufacturer’s instructions (Invitrogen #KHB3482 and #KHB3442, respectively).
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