The following reagents were purchased: mouse anti-rat GLT1/EAAT2 antibody (1:1,000, BD Transduction Laboratories), mouse anti-heterogeneous nuclear ribonucleoproteins (hnRNP)/KBBP2 antibody (1:2,000, Santa Cruz Biotechnology), rabbit anti-GLAST (EAAT1, 1:1,000, Alpha Diagnostic Intl), rabbit anti-EAAC1: neuronal glutamate transporter (EAAT3, 1:1,000, BD Transduction Laboratories); Rabbit anti-gamma-aminobutyric acid (GABA) Transporter 3 (GAT3, 1:1,000, EMD Millipore), rabbit anti-phospho-44/42 MAP kinase (Thr202/Thr204, 1:1,000, Cell Signaling), mouse anti-glial fibrillary acidic protein (GFAP, 1:2,000, Millipore), mouse anti-NeuN (1:1,000, Millipore), rabbit anti-GFP (1:1,000, Millipore), mouse anti-nitrotyrosine antibody (1:1,000, EMD Millipore), mouse anti-β-actin (1:3,000, Sigma-Aldrich), PD98059 (Mol wt: 267.28, Calbiochem), dihydrokainic acid (DHK, Mol wt: 215.25, Sigma-Aldrich), and morphine sulphate (Mol Wt: 758.83, Sigma-Aldrich). The drugs were dissolved in saline or 5% dimethyl sulfoxide (Sigma) and saline. The drug vehicle was used as a control. The doses of all agents were selected based on previous use in relevant studies.
Dihydrokainic acid dhk
Manufactured by Merck Group
Sourced in United States
Dihydrokainic acid (DHK) is a chemical compound commonly used in laboratory settings. It is a structural analog of the neurotransmitter glutamate and functions as a selective antagonist of the excitatory amino acid transporter 2 (EAAT2). DHK is employed in research applications to study the role of glutamate transporters in various biological processes.
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Lab products found in correlation
Rabbit anti gfp, by Merck Group (1 mentions)
Mouse anti neun, by Merck Group (1 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (1 mentions)
Mouse anti β actin, by Merck Group (1 mentions)
Morphine sulphate, by Merck Group (1 mentions)
Mouse anti glial fibrillary acidic protein gfap, by Merck Group (1 mentions)
Gabapentin, by Merck Group (1 mentions)
Glutamic acid, by Merck Group (1 mentions)
Enzychrom glutamate assay kit, by BioAssay Systems (1 mentions)
Hank s balanced salt solution, by Thermo Fisher Scientific (1 mentions)
Imark microplate reader, by Bio-Rad (1 mentions)
3 protocols using dihydrokainic acid dhk
1
Glutamate Transporter Immunodetection in Rodents
2
Preparation and Administration of LDN-212320
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LDN-212320 (Axon Medchem, Reston, VA, USA) was dissolved in normal saline (0.9% NaCl) with 1% dimethyl sulphoxide (DMSO) and 0.5% Tween-80 (vehicle). The solution composition for the LDN was selected based on the reported solubility and stability of this compound. We have previously reported this composition elsewhere [26 (link)].
Dihydrokainic acid (DHK) and gabapentin (positive control), purchased from Sigma-Aldrich (St. Louis, MO, USA), were dissolved in normal saline (0.9% NaCl). All control groups received an equal volume of vehicles. All drugs and chemicals were injected intraperitoneally (i.p.) at a volume of 10 mL/kg body weight unless otherwise indicated. The LDN-212320 and DHK injections were administered 24 h and 0.5 h prior to the behavioral experiments, respectively (Figure 1 ). The LDN-212320, DHK, and gabapentin doses were selected based on previous studies [41 (link),42 (link),43 (link)].
Dihydrokainic acid (DHK) and gabapentin (positive control), purchased from Sigma-Aldrich (St. Louis, MO, USA), were dissolved in normal saline (0.9% NaCl). All control groups received an equal volume of vehicles. All drugs and chemicals were injected intraperitoneally (i.p.) at a volume of 10 mL/kg body weight unless otherwise indicated. The LDN-212320 and DHK injections were administered 24 h and 0.5 h prior to the behavioral experiments, respectively (
3
Glutamate Uptake in hESC-Derived Astrocytes
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Glutamate uptake capability of the cells was measured in 28-day differentiated hESC-derived astrocytes. Glutamic acid (0.5 mM; Sigma) in Hanks’ Balanced Salt Solution (Gibco) was added to 1 × 106 cells/ml. After 0, 10, 30, 60 and 120 min, the solution was aspirated and kept at 4 °C until further testing. Human astrocytes derived from the spinal cord (from Thermo Scientific) served as positive control, while 0.5 mM Glutamic acid kept at 37 °C for 120 min served as negative control. In addition, 0.5 mM Glutamic acid kept at 4 °C for 120 min served as time 0 concentration control. The EnzyChrom™ Glutamate Assay Kit (BioAssay Systems) was used to measure the concentration of glutamate in the collected samples according to the manufacturer’s protocol and recommendations. The optical density was read at 565 nm using the iMark Microplate reader (Bio Rad). Dihydrokainic acid (DHK, 500 μM; Sigma) or 1 μM WAY-213,613 (Sigma) were used as inhibitors of GLT-1.
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