1 × 105 NCRM-1 astrocytes were plated per well of a 24-well plate and treated with TNFα or IL-1β as described. To test for glutamate uptake in astrocyte cultures, the media was removed and astrocytes equilibrated in pre-warmed Hanks balanced salt solution (HBSS) for 10 min at 37 °C, 5% CO2. Astrocytes were then incubated for 1 h at 37 °C, 5% CO2 in HBSS/0.1 mM L-glutamate (Abcam). Fifty microliters of HBSS/L-glutamate was then removed and the glutamate content determined using a Glutamate Assay Kit (Abcam) according to the manufacturer’s instructions. Glutamate uptake data were collected from three biological replicates.
L glutamate
Manufactured by Abcam
Sourced in United Kingdom
L-glutamate is an amino acid that plays a crucial role in various cellular processes. It is a key component of the glutamate-glutamine cycle and is involved in neurotransmission, energy metabolism, and protein synthesis.
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2 protocols using l glutamate
1
Quantifying Astrocyte Glutamate Uptake
2
Pharmacological Modulation of Neuronal Signaling
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L-glutamate (Abcam, Cambridge, UK) was dissolved in 1 molar eq. NaOH and used at a final concentration of 1 mM. Excitatory glutamate agonists NMDA (Tocris, Bristol, UK), kainate (Tocris) and AMPA (Abcam) were dissolved in dH 2 O and used at 50 µM. The MEK1/2 inhibitor PD184352 (Sigma, St. Louis, MO, USA), CaMKII inhibitor KN-62 (Cayman Chemical, Michigan, USA), PI3K inhibitor LY294002 (Tocris) and the NMDAR inhibitor D-AP5 (Tocris) were dissolved in DMSO and used at 2 µM, 10 µM, 10 µM and 50 µM, respectively. The cell-permeable phosphopeptide activator of PI3K, 740 Y-P (Tocris), was dissolved in dH 2 O and used at 10 µg/ml. The Na + channel blocker tetrodotoxin (TTX) citrate (Abcam) was dissolved in dH 2 O and used at 2.5 µM. DMSO or NaOH at the corresponding concentrations were used as vehicle for control conditions.
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