4-Hydroxynonenal (4-HNE) levels were measured using the ELISA method. Samples were homogenized and centrifuged to obtain total protein. 5 ug of protein was added to each well on a protein binding plate (28298-602; VWR, Ville Mont-Royal, Quebec, Canada) and incubated overnight at 4 °C. With washes between each step (5×), blocking buffer (5% BSA) was added for 1 h at room temperature (RT), followed by the primary antibody (393207, EMD Millipore) for 1 h at RT. Secondary antibody conjugated to horse radish peroxidase (7074; Cell Signaling Technology, Danvers, MA, USA) was added for 1 h at RT. TMB substrate solution was added for 20 min followed by stop solution (2M HCl). Absorbance was measured at 450 nm with 620 nm as a reference using a microplate reader (BioTek® Instruments). The absorbance was compared against that of a standard curve using known amounts of 4-HNE created by mixing 4-HNE (A8806; Sigma–Aldrich, Oakville, ON, Canada) with fatty acid free BSA (A7030; Sigma–Aldrich) as previously described (Weber et al., 2013 (link)).
A8806
Manufactured by Merck Group
Sourced in United States
A8806 is a laboratory equipment product manufactured by Merck Group. It is a precision instrument designed for scientific applications, but a detailed description of its core function cannot be provided while maintaining an unbiased and factual approach. Further information is not available.
Automatically generated - may contain errors
Lab products found in correlation
O1008, by Merck Group (3 mentions)
F6428, by Merck Group (3 mentions)
P0500, by Merck Group (3 mentions)
Superfrost plus glass slide, by Thermo Fisher Scientific (2 mentions)
Inverted sp5x confocal microscope system, by Leica (2 mentions)
P1383, by Merck Group (2 mentions)
P9767, by Merck Group (2 mentions)
G7793, by Merck Group (2 mentions)
A7030, by Merck Group (1 mentions)
L3771, by Merck Group (1 mentions)
M9272, by Merck Group (1 mentions)
Lauric acid, by Merck Group (1 mentions)
0.22 mm low protein binding filter, by Merck Group (1 mentions)
C0832, by Merck Group (1 mentions)
Dmem no phenol red, by Thermo Fisher Scientific (1 mentions)
Triacsin c, by Bio-Techne (1 mentions)
Mouse rat leptin elisa, by ALPCO (1 mentions)
Dulbecco phosphate buffered saline (dpbs), by Thermo Fisher Scientific (1 mentions)
Dulbecco s phosphate buffered saline dpbs, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Gemini Bio (1 mentions)
45 protocols using a8806
1
Quantifying 4-Hydroxynonenal Levels
2
Lipid Extraction and Analysis Protocol
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The liver lipids (0.5 g) were extracted using the Folch method52 (link). An aliquot of the extract was subjected to gravimetric analysis to measure the total lipid concentrations. The remaining extract was allowed to evaporate under a nitrogen flow and dissolved in 1 mL of lipoprotein lipase buffer, containing 1,4-Piperazinediethanesulfonic acid disodium salt ((P3768, Sigma-Aldrich), MgCl2·6 H2O (M9272, Sigma-Aldrich), albumin-free fatty acids (A8806, Sigma-Aldrich) and 0.1% sodium dodecyl sulfate (L3771, Sigma-Aldrich). The triglycerides and cholesterol concentrations in the dissolved extract and in the cultured cell lysates were measured using QCA enzymatic colorimetric kits (QCA, Barcelona, Spain) according to the manufacturer’s protocols.
3
Fatty Acid Albumin Complex Protocols
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A 12.5% fatty acid-free bovine serum albumin (A8806, Sigma, hereafter BSA) was prepared in water and subsequently sterile filtered. This solution was diluted to either 10% BSA using sterile filter 55 mM NaOH solution, aliquoted and stored at −20 °C until use, or further complexed with OA. For this, OA (O1008, Sigma) was dissolved in 55 mM NaOH at a final concentration of 50 mM and kept at 65 °C with frequent vortex to allow dissolution. Once dissolved, this OA solution was quickly sterile filter and then added to the 12.5% BSA solution to reach the final concentration of 10 mM OA in 10% BSA. Aliquots were then kept at −20 °C until further use.
Proliferating SVZ NSPCs were incubated with the appropriate concentration of BSA (0.1% or 0.5%) or OA (0.1 mM or 0.5 mM) for 15 h at 37 °C. Thereafter, they were fixed for immunohistochemistry or were subsequently plated under differentiation condition (see “Cell culture”) and fixed at day 7. For the differentiation experiments including the wash steps, NSPCs were collected after the 15 h of loading/efflux, spun down (300 × g, 3 min) and the pellet was washed 2× with PBS. These washed NSPCs were then plated under differentiation condition into fresh plates and fixed at day 7.
Proliferating SVZ NSPCs were incubated with the appropriate concentration of BSA (0.1% or 0.5%) or OA (0.1 mM or 0.5 mM) for 15 h at 37 °C. Thereafter, they were fixed for immunohistochemistry or were subsequently plated under differentiation condition (see “Cell culture”) and fixed at day 7. For the differentiation experiments including the wash steps, NSPCs were collected after the 15 h of loading/efflux, spun down (300 × g, 3 min) and the pellet was washed 2× with PBS. These washed NSPCs were then plated under differentiation condition into fresh plates and fixed at day 7.
4
Preparation of Palmitate and Laurate Solutions
5
Fatty Acid Conjugation and Treatment
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All fatty acid treatments were done using FFAs conjugated to BSA (fatty acid and endotoxin free, A8806, Sigma). The conjugation was performed by preparing a sterile-filtered 5% BSA solution in macrophage differentiation medium. Both 5% BSA medium and concentrated fatty acid solution (100 mM of fatty acid in ethanol) were heated at 60°C before adding fatty acid solution dropwise into 5% BSA medium in order to make a medium containing 2.5 mM fatty acid and 5% BSA (approximately 10:3 fatty acid to BSA molar ratio). This medium was then sonicated until it became completely clear and used as a stock solution for stimulations on the same day without sterile filtering. FFA-free BSA solution with equivalent amount of ethanol was used as a control. In dose-response experiments, the amount of BSA and ethanol in each condition was adjusted to the highest dose of palmitate. Unless otherwise indicated, fatty acid treatments were performed overnight for 16 hr.
6
Adipose Tissue Lipolysis Assay
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We used a modified version of a well-established lipolysis assay [36] (link) to evaluate NEFA, glycerol, and leptin release from adipose organ explants. Briefly, eWAT, iWAT, and BAT from Adipoq-GsD, Adipoq-GiD and respective littermate controls were surgically removed and cut in ∼20 mg pieces. The pieces were put in a 96-well plate containing 200 μl of prewarmed (37 °C) DMEM no phenol red (Gibco A1443001). The pieces were then incubated at 37 °C (5% CO2, and 95% humidified atmosphere) for one hour (baseline) in DMEM containing 2% fatty acid (FA)-free bovine serum albumin (BSA, Sigma, A8806) and 0.1% glucose (Sigma 49163). To avoid re-esterification of FA and glycerol, each well also contained 5 μM of the acyl-CoA synthetases inhibitor triacsin C (Tocris 2472). At the end of the hour, the pieces were transferred to a new 96-well plate containing fresh medium with 1 μM CNO (C0832, Sigma) and incubated for another hour (stimulated). The incubation media from baseline and stimulated conditions were then used to evaluate NEFA release by colorimetry (FUJIFILM Wako Diagnostics-NEFA Reagent, 999-34691, 995-34791, 991-34891, 993-35191), glycerol release by colorimetry (F6428, Sigma), and leptin release by ELISA (Mouse/Rat Leptin ELISA, ALPCO, 22-LEPMS-E01). NEFA, glycerol, and leptin release were then calculated based as a percentage compared to baseline.
7
Preparation of Advanced Glycation End-products
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AGE was prepared according to methods described previously [11 (link),39 (link),40 (link)]. Briefly, AGE was prepared by incubating bovine serum albumin (A8806, BSA, fraction V, fatty acid-free, endotoxin-free; Sigma Chemical, 100 mg/mL) with D-glucose (1 M) in Dulbecco’s phosphate-buffered saline (DPBS, 21600–010; Gibco) for 12 weeks at 37 °C. Unmodified BSA was prepared under the same conditions without glucose as a control. The fluorescence of the supernatant was determined (excitation 370 nm, emission 440 nm) using LJL Biosystems (AnalystTM AD, Sunnyvale, CA, USA), which confirmed the higher intensity of AGEs in AGE-modified BSA than in unmodified BSA in our previous paper [21 (link)]. The AGE solution was filter sterilized by a 0.8 μM Millex GP filter unit (Millipore, Billerica, MA, USA).
8
Imaging-Based Assays with dHL-60 Cells
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Imaging-based assays with dHL-60 cells were performed using 96-well #1.5 glass-bottom plates (Brooks Life Sciences). The wells were coated with a 100 µl solution of 10 µg/ml porcine fibronectin (prepared from whole blood) and 0.4 mg/ml bovine serum albumin (BSA; endotoxin-free, fatty acid free; A8806; Sigma) dissolved in Dulbecco’s phosphate-buffered saline (DPBS; Life Technologies) and incubated for 30 min at RT. Fibronectin solution was then aspirated, and each well was washed twice with DPBS. dHL-60 cells in growth media were pelleted at 300 × g for 5 min, resuspended in 100 µl imaging media (RPMI1640 with 0.2% BSA), plated in each well and incubated (37°C/5% CO2) for 20 min for cells to adhere. Primary human neutrophils were also plated as above and processed similarly as the dHL-60 cells for immunofluorescence assays.
9
Oleate Uptake in U-2 OS Cells
10
Immunofluorescence Imaging of Cells
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Cells grown over Matrigel‐coated coverslips were fixed with 4% paraformaldehyde (SantaCruz Biotechnology #SC‐281692), permeabilized with 0.1% Triton X‐100 (Sigma‐Aldrich #T8787), blocked in PBS (ThermoFisher Scientific #A12856‐01) with 1% bovine serum albumin (BSA, Sigma‐Aldrich #A8806) for 30 minutes at 37°C and incubated with the primary antibody overnight at 4°C. Cells were subsequently incubated with the secondary antibody for 30 minutes at 37°C and mounted with ProLong Gold Antifade Mountant with 4′,6‐diamidino‐2‐phenylindole (DAPI, ThermoFisher Scientific #P36930). Primary and secondary antibodies are listed in Table S1 . For Ki‐67 detection, we first performed an antigen retrieval step in which cells were heated for 10 seconds in a microwave with citrate buffer pH 6.0 (Sigma‐Aldrich #C9999) letting cells cool down 20 minutes. Acquisition of fluorescence images was performed in a Leica TCS‐SP5 or a Nikon Eclipse Ti fluorescence microscope. Images were processed using the Adobe Photoshop CS5 or ImageJ software.28 Positive cells were counted using the ImageJ software from at least three random fields per preparation.
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