The MDA content was measured using a TBARS assay kit (10009055, Cayman Chemical) according to manufacturer's protocol. Briefly, HUVECs after oxLDL and/or PD treatment for 24 hrs were lyzed in PBST (106 cells in 0.1 mL PBST) by sonication on ice. 100 μL of cell lysate or standard was added to a 5 mL vial, which was mixed in 100 μL of SDS solution and 4 mL of the color reagent. The vials were capped and boiled for 60 min, incubated on ice for 10 min, and centrifuged 10 min at 1600 ×g at 4°C. 150 μL of supernatant was transferred into an assay plate and read on a FLx800 fluorescent reader (BioTek) with excitation 530 nm and emission 550 nm.
Flx800 fluorescent reader
Manufactured by Agilent Technologies
The FLx800 fluorescent reader is a laboratory instrument designed for the detection and measurement of fluorescent signals. It is a versatile tool used for a variety of applications that require fluorescence-based analysis.
Automatically generated - may contain errors
Lab products found in correlation
3 protocols using flx800 fluorescent reader
1
Measurement of Lipid Peroxidation in HUVECs
2
Cellular Uptake of Cholesteroyl Hyaluronic Acid Nanogels
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MiaPaCa-2 cells were seeded in 24-wells
plates. Rhodamine-labeled HA and CHA nanogel (50 μg/mL; cholesterol
contents 6, 10, and 18 moieties per polymer molecule) were incubated
with cells in full medium for 2 h at 4 or 37 °C in triplicates.
Cells were collected, washed 3 times with ice cold PBS, and treated
with cell lysis buffer (100 μL) for 10 min. Each sample was
transferred into black-walled plate, and fluorescence was measured
using a BioTek FLx-800 fluorescent reader.
To investigate the
uptake pathways for CHA nanogels, we used endocytosis pathway inhibitors.22 (link) Uptake of Rh-CHA in MiaPaCa-2 cells was measured
after pretreatment with free HA (2 mg/mL) to block CD44 receptors,
using low temperature (4 °C) to inhibit endocytosis, in the presence
of 10 μg/mL chlorpromazine (clathrin-dependent endocytosis inhibitor),
10 μg/mL genistein (caveolae-dependent endocytosis inhibitor),
or 100 μg/mL amiloride (macropinocytosis inhibitor). The internalized
nanogels were quantified by fluorescence using calibration curves.
plates. Rhodamine-labeled HA and CHA nanogel (50 μg/mL; cholesterol
contents 6, 10, and 18 moieties per polymer molecule) were incubated
with cells in full medium for 2 h at 4 or 37 °C in triplicates.
Cells were collected, washed 3 times with ice cold PBS, and treated
with cell lysis buffer (100 μL) for 10 min. Each sample was
transferred into black-walled plate, and fluorescence was measured
using a BioTek FLx-800 fluorescent reader.
To investigate the
uptake pathways for CHA nanogels, we used endocytosis pathway inhibitors.22 (link) Uptake of Rh-CHA in MiaPaCa-2 cells was measured
after pretreatment with free HA (2 mg/mL) to block CD44 receptors,
using low temperature (4 °C) to inhibit endocytosis, in the presence
of 10 μg/mL chlorpromazine (clathrin-dependent endocytosis inhibitor),
10 μg/mL genistein (caveolae-dependent endocytosis inhibitor),
or 100 μg/mL amiloride (macropinocytosis inhibitor). The internalized
nanogels were quantified by fluorescence using calibration curves.
3
Plasma Antioxidant Capacity by FRAP and ORAC
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Antioxidant capacity of plasma was determined by the FRAP assay in a redox-linked colorimetric reaction using methods modified from Benzie and Strain [28 (link)]. Plasma was incubated at room temperature with the FRAP reagent and absorbance recorded on a Synergy Analyzer (Bio Tek Instruments) at a wavelength of 593 for 4 minutes. Trolox was used as standard and the results are expressed mmol Trolox equivalents (TE) per liter.
Plasma samples were analyzed for hydrophilic and lipophilic ORAC according to published methods [29 (link),30 (link)]. The assay was carried on 48-well microplates using the FLX 800 fluorescent reader (Bio Tek Instruments) with an excitation wave-length of 546 nm and an emission wavelength of 565 nm and the reaction was monitored for 1 hour and 15 minutes. Calculations were made using Microsoft Excel and the data expressed as mmol TE per liter.
Plasma samples were analyzed for hydrophilic and lipophilic ORAC according to published methods [29 (link),30 (link)]. The assay was carried on 48-well microplates using the FLX 800 fluorescent reader (Bio Tek Instruments) with an excitation wave-length of 546 nm and an emission wavelength of 565 nm and the reaction was monitored for 1 hour and 15 minutes. Calculations were made using Microsoft Excel and the data expressed as mmol TE per liter.
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