Ldh release assay

Since a larger quantity of cortical neurons can be obtained from one embryo compared to hippocampal neurons, cortical neurons were used for NMDA excitotoxicity assay. Cortical neurons at 11–12 days in vitro (DIV) were pre-treated with PSR or vehicle (DMSO) for 2 h and subsequently rinsed with Locke’s solution (5 mM KCl, 128 mM NaCl, 2.7 mM CaCl₂, 1 mM Na₂HPO₄, 5 mM HEPES, and 10 mM glucose) followed by a 15-min incubation with glycine-containing (10 μM) Locke’s solution. The neurons were then co-treated with PSR (or vehicle) and 20 μM NMDA dissolved in Locke’s plus glycine solution for 20 min. The neurons were then incubated with fresh growth medium. After 24 h, NMDA excitotoxicity was assessed by either the lactate dehydrogenase (LDH) release assay (Roche) or immunohistochemistry, as described previously [52 (link)]. The cell death in the presence of PSR and NMDA was quantified by normalizing the LDH release to the maximum (vehicle with NMDA, the control) and minimum (no NMDA) LDH releases. Neurons were immunostained with β-tubulin type III antibody (1:1000, Sigma) and labeled with FITC-conjugated secondary antibody. DAPI (4′, 6-diamidino-2-phenylindole) was used to stain the nuclei. Images were captured and visualized by fluorescent microscopy (Leica).

CPE species-treated confluent parent cells, claudin-4 transfectant cells, or Caco-2 cells were collected by centrifugation at 2,000 × g for 3 min, and supernatants from those centrifugations were applied to fresh confluent parent cell cultures for 1 h at 37°C. Cytotoxicity in these cultures was measured by microscopy (as described above) or by using an MTT assay kit (CellTiter 96 Aqueous one-solution cell proliferation assay; Promega) or LDH release assay (Roche) per the manufacturer’s instructions. LDH release specifically induced by supernatant treatment of parent cells was calculated after subtracting the LDH already present in the supernatants due to its release from CPE-treated Caco-2 cells or claudin-4 transfectants.