After 4 h of Aβ1–42 intoxication (Supplementary Table 1 ), cell media supernatants were analysed with Amplex Red Glutamic Acid assay kit (Invitrogen) according to the manufacturer's instructions.
Amplex red glutamic acid assay kit
Manufactured by Thermo Fisher Scientific
Sourced in United States
The AMPLEX Red glutamic acid assay kit is a fluorometric assay kit designed to measure glutamic acid levels in various samples. The kit utilizes the AMPLEX Red reagent, which reacts with glutamic acid to produce a fluorescent product that can be detected and quantified.
Automatically generated - may contain errors
Lab products found in correlation
Fetmpyp, by Cayman Chemical (2 mentions)
Fetmpyp, by Merck Group (2 mentions)
Opti mem, by Thermo Fisher Scientific (2 mentions)
Shikonin, by Merck Group (1 mentions)
Fluostar omega microplate reader, by BMG Labtech (1 mentions)
Bca bicinchoninic acid assay kit, by Nanjing Jiancheng (1 mentions)
Glutamic acid assay kit, by Nanjing Jiancheng (1 mentions)
10 protocols using amplex red glutamic acid assay kit
1
Aβ1-42 Intoxication Glutamic Acid Assay
2
Measuring Glutamate Release in Cells
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Shikonin (Sigma, S7576) was dissolved in DMSO at a concentration of 34.8 mM, then diluted to a final concentration of 20 µM in a suspension of cells (300 000 cells ml−1) in Hank's Balanced Salt Solution (HBSS). After 90 min of incubation at 37°C, the cells and cell fragments were spun down and glutamate concentration was measured in the supernatant, using an Amplex Red glutamic acid assay kit (Invitrogen) and a Fluostar Omega microplate reader (BMG Labtech, Ortenberg, Germany), with excitation at 544 nm and fluorescence detection at 590 nm. A second-order polynomial function was fitted by least squares to measurements from known glutamate standard solutions, and inverted to yield glutamate measurements from the fluorescence intensity measurements.
3
Glutamate Quantification in ALS Astrocytes
4
Glutamate Uptake Assay in Ischemic Cortex
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The ischemic hemisphere cortex tissues and cell supernatants were prepared after treating with varying experimental conditions. For data from in vivo, the cortex was isolated from the ischemic hemisphere of mouse.
After homogenizing the cortex, glutamate uptake was determined by Glutamic Acid assay kit (Nanjing Jiancheng, China) according to the manufacturer's protocols. Protein concentration was estimated by Bradford assay using the BCA (Bicinchoninic acid) assay kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China).
For data from in vitro, well plates, and the glutamate uptake was measured using the Amplex Red Glutamic Acid assay kit (Invitrogen Life Technolog ies Inc., Grand Island, NY, USA). After 6 h of OGD astrocyte culture medium was replaced by Hepes buffer containing 25 mM glucose and 500 μM glutamate. After 24 h incubation, 50 μL of supernatants was transferred into 96-well plates, and then mixed with 50 μL substrate mixture and incubated at 37 for 30 min. Fluorescence was measured using an automated microplate reader at a wavelength of 530 nm (vs. reference wavelength of 590 nm). Glutamate concentrations were calculated from the standard curve with known glutamate amounts.
After homogenizing the cortex, glutamate uptake was determined by Glutamic Acid assay kit (Nanjing Jiancheng, China) according to the manufacturer's protocols. Protein concentration was estimated by Bradford assay using the BCA (Bicinchoninic acid) assay kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China).
For data from in vitro, well plates, and the glutamate uptake was measured using the Amplex Red Glutamic Acid assay kit (Invitrogen Life Technolog ies Inc., Grand Island, NY, USA). After 6 h of OGD astrocyte culture medium was replaced by Hepes buffer containing 25 mM glucose and 500 μM glutamate. After 24 h incubation, 50 μL of supernatants was transferred into 96-well plates, and then mixed with 50 μL substrate mixture and incubated at 37 for 30 min. Fluorescence was measured using an automated microplate reader at a wavelength of 530 nm (vs. reference wavelength of 590 nm). Glutamate concentrations were calculated from the standard curve with known glutamate amounts.
5
Quantifying Glutamate Release in Cells
6
Serum Glutamate Quantification Protocol
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Serum proteins were precipitated from samples using 1 volume of chilled 100% (w/v) trichloroacetic acid (TCA) to 3 volumes of serum. Samples were centrifuged and the supernatant removed and neutralized with potassium hydroxide (KOH). Protein pellets were quantified for each sample using the BioRad assay as described in “Supplementary Materials and Methods”. Serum glutamate in the supernatant was analyzed using the AMPLEX Red glutamic acid assay kit (Invitrogen/Molecular Probes, Eugene, OR, USA) and analyzed on a CytoFluor Series 4000 Fluorescence Multi-Well Plate Reader (PerSeptive Biosystems, Framingham, MA, USA). Serum glutamate was normalized to total serum protein for each sample.
7
Quantifying Glutamate Release in Cell Cultures
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Immediately prior to 14C-cystine uptake quantification, growth media samples from 6-well plates were collected and stored at 4°C. Glutamate release was quantified using the AMPLEX Red glutamic acid assay kit (Invitrogen/Molecular Probes, Eugene, OR). This assay quantitates the fluorescent reaction product resorufin, which is produced in proportion to glutamate, for analysis on a CytoFluor Series, 4000 Fluorescence Multi-Well Plate Reader (PerSeptive Biosystems, Framingham, MA). To optimize this assay for measurement of media glutamate concentrations beyond 0.5 µM and eliminate the repeated cycling of glutamate and α-ketoglutarate, L-alanine and L-glutamate pyruvate transaminase were omitted from the reaction mixture. 37 (link) Background glutamate from a media-only control well was subtracted from extracellular glutamate levels in culture media, and values were normalized to total protein using the Bradford protein assay, as mentioned earlier.
8
Femur Glutamate Quantification
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Animals were killed on day 10 or day 14, and both femurs were removed. Proximal and distal ends of the femur were clipped and the intramedullary contents rinsed into a vial with 0.1M PBS containing protease inhibitor cocktail and EDTA (Pierce, Rockford, IL). Femur contents from 2 to 4 animals per treatment were pooled per sample. AMPLEX Red glutamic acid assay kit (Invitrogen/Molecular Probes) was used to determine glutamate concentration from pooled samples.
9
Glutamate Release Modulation by Antioxidants
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66.1 cells were seeded on 12-well plates and pretreated for 2 hours with Fe(III) meso-tetrakis (N-methylpyridinium-4-yl)porphyrin chloride (FeTMPyP; Cayman Chemical, Ann Arbor, MI), SSZ (Sigma-Aldrich, St. Louis, MO), Mn(III)-5,15-di-phenyl-tetracyclohexenylporphyin chloride (SRI10), or vehicle-containing (0.01 or 0.02% DMSO) MEM and then 6 hours with 3-morpholinosydnonimine (SIN-1) with and without FeTMPyP, SSZ, or SRI10. Cells were then incubated in a drug-containing, l -glutamine-, phenol red-, and serum-free Opti-MEM (Life Technologies, Carlsbad, CA) for 18 hours. Media was then harvested for glutamate analysis while cells were trypsinized and counted. Glutamate release was quantified with the AMPLEX Red glutamic acid assay kit (Invitrogen/Molecular Probes, Eugene, OR) that was optimized for measurement of glutamate concentrations above 0.5 μM by omitting l -alanine and l -glutamate pyruvate transaminase from the reaction, thus eliminating signal amplification through repeated cycling of glutamate through α-ketoglutarate31 (link) and analyzed on a Synergy Bio-Tek Plate Reader.
10
Femur Glutamate Concentration Analysis
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