Glutamate excitotoxicity

In primary CX3CR1^+/+ (WT) and CX3CR1^−/− hippocampal cultures, the conditioned medium was removed and stored for later use; neurons were washed and treated with glutamate (50 μM glutamate + 10 μM glycine, 30 min) in modified Locke’s buffer (without MgCl₂ plus 1 μM glycine) to stimulate all types of glutamate receptors. When necessary, cells were pre-treated with polyclonal CX3CR1 Ab (10 μg/ml, 30 min; sc-30030, Santa Cruz, Dallas, TX, USA) or rabbit IgG (10 μg/ml, 30 min; BA-1000, Vector laboratories, Burlingame, CA, USA) in culture medium; drugs were present during and after the glutamate challenge. After treatment, cells were re-incubated in the original conditioned medium for 18–20 hours.

Glutamate excitotoxicity towards neurons was measured using the lactate dehydrogenase (LDH)-based Cyto Tox 96 non-radioactive cytotoxicity assay kit (TOX7, Sigma-Aldrich, St. Louis, MO, USA) in accordance with the manufacturer’s protocol. Glutamate-treated cultured neurons were pelleted by centrifugation at 250 × g for 4 min. The supernatant was transferred to a new 96-well plate, and the reconstituted substrate mix was added. The absorbance was measured at 490 nm. Each assay was tested in triplicate. The percentage of specific lysis was determined as follows: (experimental release-target spontaneous release)/ (target maximum release-target spontaneous release) × 100. The maximum release was determined by detecting the absorbance of the target cells lysed with LDH assay lysis solution.

The viability of glutamate-treated cultured neurons was tested by an MTT assay. Specifically, 5 mg/ml MTT was added 1:10 to the cultures and incubated for 2 hours; the medium was aspired, then the cells were treated with DMSO and incubated at 37 °C for 10 min. Samples were then analyzed with a microplate reader at 490 and 630 nm to subtract the background. The results were expressed as a percentage (%) of viable cells in the control cultures.