The concentration of intracellular glutamate was detected using the Amplex®Red Glutamic Acid / Glutamate Oxidase Assay Kit (Invitrogen, USA) according to the manufacture′s procedure. TGF-β1-treated cells were lysed by RIPA buffer supplemented with protease inhibitor cocktail. 50 μl of supernatant or H2O2 positive control was diluted by 1 × reaction buffer, then 50 μl of Amplex®Red reagent / HRP / glutamate oxidase / glutamate—pyruvate transaminase / alanine working solution was added to start the reaction. The sample was incubated at 37°C for 30 min in darkness. The plate was measured at 530 / 590 nm to calculate the concentration of intracellular glutamate.
Amplex red glutamic acid glutamate oxidase assay kit
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Amplex Red Glutamic Acid/Glutamate Oxidase Assay kit is a fluorometric detection kit that measures glutamic acid and glutamate levels in various sample types. The kit uses the Amplex Red reagent and glutamate oxidase to produce a fluorescent product, which can be detected using a fluorescence microplate reader.
Automatically generated - may contain errors
Lab products found in correlation
96 well assay plate, by Corning (2 mentions)
Synergy neo2, by Agilent Technologies (2 mentions)
Flexstation 3, by Molecular Devices (1 mentions)
Lncap psma atcc crl 1740, by American Type Culture Collection (1 mentions)
Rabbit anti rbp4 antibody, by Abcam (1 mentions)
Glutamate assay kit, by Merck Group (1 mentions)
Zorbax eclipse aaa, by Agilent Technologies (1 mentions)
Celltiter glo luminescent cell viability assay, by Promega (1 mentions)
Rat tnf α elisa kit, by Thermo Fisher Scientific (1 mentions)
Mitotracker green fm dye, by Thermo Fisher Scientific (1 mentions)
Mitosox red, by Thermo Fisher Scientific (1 mentions)
Versene, by Thermo Fisher Scientific (1 mentions)
Mitotracker orange cm h2tmros, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (1 mentions)
Astrocyte medium, by ScienCell (1 mentions)
Synergy h1 hybrid multi mode microplate reader, by Agilent Technologies (1 mentions)
Dl threo β benzyloxyaspartate tboa, by Bio-Techne (1 mentions)
Spectramax m4, by Molecular Devices (1 mentions)
Kinase glo luminescent kinase assay, by Promega (1 mentions)
Methyl thiazolyl tetrazolium (mtt), by Merck Group (1 mentions)
47 protocols using amplex red glutamic acid glutamate oxidase assay kit
1
Intracellular Glutamate Quantification
2
Amyloid-Beta Induced Glutamate Release
3
Quantification of Glutamate Levels
4
L-Phenylalanine Oxidase Activity Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Peroxide was determined by a method based on the commercial Amplex® Red Glutamic Acid/Glutamate Oxidase Assay Kit (Invitrogen). The reaction mixture contained 25 mM L-phenylalanine, 10 μM pvLAAD protein and with or without 2 mg/mL of E. coli membrane. Reactions were incubated at 37 °C for 60 min. Then, working solution (100 μM Amplex® Red reagent, 0.25 U/mL HRP) was added, and the reactions were incubated at 37 °C for 40 min. Then, fluorescence emission at 585 nm (excited at 571 nm) were recorded using a microplate reader (Flex Station 3, Molecular Devices). The reactions without pvLAAD protein were used as the blank control. The standard curve of H2O2 concentrations was generated using H2O2 samples at different concentrations (5000, 2000, 1000, 500, 250, 125, 62.5 μM).
5
Prealbumin Quantification in Plasma
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Prealbumin
from human plasma
(human TTR) was purchased from Sigma (Sigma: #P1742). Amplex Red Glutamic
Acid/Glutamate Oxidase Assay Kit (Invitrogen, Fisher Scientific; Molecular
Probes, A12221). N-Acetylaspartylglutamate (NAAG; MP Biomedicals,
ICN15303625), rhPSMA (R&D Research, 4234ZN010). rhPSMA (20 μM
in reaction buffer; R&D Research, 4234ZN010), PMPA obtained from
Tocris (cat # 13–801–0). Human prostate carcinoma cell
lines LNCaP (PSMA+) (ATCC CRL-1740) and DU145 (PSMA−) (ATCC
HTB81) cell lines were obtained from American Type Culture Collection
(ATCC), Manassas, USA. Cathepsin B from human liver was purchased
from Calbiochem, EMD Millipore Corp (# 219362–50UG). Rabbit
anti-RBP4 antibody was purchased form Abcam (#ab154914). IRdye800
donkey antirabbit secondary antibody was purchased from LI-COR Biosciences
(#926–32213).
from human plasma
(human TTR) was purchased from Sigma (Sigma: #P1742). Amplex Red Glutamic
Acid/Glutamate Oxidase Assay Kit (Invitrogen, Fisher Scientific; Molecular
Probes, A12221). N-Acetylaspartylglutamate (NAAG; MP Biomedicals,
ICN15303625), rhPSMA (R&D Research, 4234ZN010). rhPSMA (20 μM
in reaction buffer; R&D Research, 4234ZN010), PMPA obtained from
Tocris (cat # 13–801–0). Human prostate carcinoma cell
lines LNCaP (PSMA+) (ATCC CRL-1740) and DU145 (PSMA−) (ATCC
HTB81) cell lines were obtained from American Type Culture Collection
(ATCC), Manassas, USA. Cathepsin B from human liver was purchased
from Calbiochem, EMD Millipore Corp (# 219362–50UG). Rabbit
anti-RBP4 antibody was purchased form Abcam (#ab154914). IRdye800
donkey antirabbit secondary antibody was purchased from LI-COR Biosciences
(#926–32213).
6
Glutamate Measurement in Cell Cultures
7
Intracellular and Extracellular Glutamate Quantification
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Intracellular glutamate detection was performed with the Amplex Red Glutamic Acid/Glutamate Oxidase Assay Kit from Invitrogen following manufacturer’s procedure. High performance liquid chromatography (HPLC) analysis for extracellular glutamate was performed as previously described [25 (link)].
8
Quantifying Glutamate Release Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The release of intracellular glutamate into the extracellular medium was quantified using the Amplex Red Glutamic Acid/Glutamate Oxidase Assay Kit (Invitrogen, A12221) following the manufacturer’s protocol. Specifically, upon completing the treatment, cells in 6-well plates were subjected to two washes with PBS and then incubated in Na+-containing and glutamine-free media containing either DMSO or erastin for 1 hour. Subsequently, 50 μl of the medium from each well was transferred to a 96-well plate and incubated with 50 μl of a reaction mixture for 30 minutes. The fluorescence intensity of each sample was then measured using a fluorescence plate reader. After normalizing the fluorescent signal to the total cell number at the end of the experiment, the amount of glutamate release was expressed as a percentage of the control group with no treatment (DMSO).
9
Quantifying Intracellular Glutamate Levels
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Glutamate levels were analyzed by RP-HPLC using an Agilent 1200 liquid chromatograph and fluorescence detector as previously described [14 (link)] with a few modifications. The experiments utilized 4.6 × 75 mm, 3.5 μm ZORBAX Eclipse AAA analytical columns (Agilent). A gradient elution program was optimized for glutamate measurement with a flow rate 0.75 ml/min. The intracellular glutamate levels in the whole brain lysates of mice and whole cell lysates were determined by Amplex Red Glutamic Acid/Glutamate Oxidase Assay Kit (Invitrogen) based on the manufacturer’s instruction. The brain tissue lysates and whole cell lysates were diluted to the same protein concentration before the assay.
10
Astroglia Glutamate Uptake Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Astroglia were plated at a concentration of 20,000 cells per well in a 48-well
plate and cultured for an additional 2 days to reach confluence. Before the
assay, cultures were equilibrated in Hank's balanced salt solution (HBSS) buffer
for 10 min.L -Glutamate (20 μM) solutions
were prepared with HBSS and incubated with cells. After 30 and
60 min, the glutamate concentration remaining in the medium was measured
using Amplex Red Glutamic Acid/Glutamate
Oxidase Assay Kit (Invitrogen)
according to the manufacturer’s instructions. The decreased
glutamate was reported as
micromoles of glutamate per
micrograms of protein after being normalized to the total protein in each well.
The protein concentration was measured by Micro
BCA protein assay kit (Pierce).
plate and cultured for an additional 2 days to reach confluence. Before the
assay, cultures were equilibrated in Hank's balanced salt solution (HBSS) buffer
for 10 min.
were prepared with HBSS and incubated with cells. After 30 and
60 min, the glutamate concentration remaining in the medium was measured
using Amplex Red Glutamic Acid/Glutamate
Oxidase Assay Kit (Invitrogen)
according to the manufacturer’s instructions. The decreased
glutamate was reported as
micromoles of glutamate per
micrograms of protein after being normalized to the total protein in each well.
The protein concentration was measured by Micro
BCA protein assay kit (Pierce).
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!