Materials and Reagents

1. 96 well plate, flat bottom (SARSTEDT, catalog number: 82.1581 )
   
2. Pipette tips
   
3. Neuronal cell cultures which was treated by noxes (e.g., oxygen and glucose deprivation, glutamate or other toxins) (Koh and Choi, 1987; Bruer et al., 1997; Harms et al., 2001; Ruscher et al., 2002; Harms et al., 2004; Meisel et al., 2006; Harms et al., 2007; Datwyler et al., 2011; Schweizer et al., 2015; Donath et al., 2016)
   Note: This protocol works for primary neuronal cultures that were derived from various regions of the brain including septum, hippocampus, striatum, spinal cord, cortex, cerebellum or raphe nuclei and that were seeded in various densities or with various media formulations or coatings of the plates. However, we achieve best results with cultures that are at least cultivated for more than 7 days in vitro (DIV) and that were seeded in a reasonable density as illustrated below (Figures 1 and 2). These cultures contain approximately 10% glial cells and this does not impact on the possibility to analyze LDH release in the supernatant medium.
   
   
   Figure 1. Primary cortical neuronal cultures derived from mouse E15 embryos after 7 days in vitro (DIV). Scale bar = 50 µm.
   
   
   Figure 2. Primary cortical neuronal cultures that were treated with 50 µM glutamate for 24 h on DIV 8. Scale bar = 50 µm.
   
   
4. Triton X-100 solution (Sigma-Aldrich, catalog number: 93443 )
   
5. Potassium phosphate monobasic (KH₂PO₄) (MW 136.1) (Sigma-Aldrich, catalog number: P5655 )
   
6. Dibasic potassium phosphate (K₂HPO₄; MW 174.2) or potassium phosphate trihydrate (K₂HPO₄·3H₂O; MW 228.2) (Sigma-Aldrich catalog number: P5504 )
   
7. Distilled water
   
8. Na-pyruvate (MW 110) (Sigma-Aldrich, catalog number: P2256 )
   
9. LDH standard (TruCal U) (DiaDys Diagnostic Systems, catalog number: 5 9100 99 10 063 )
   
10. -NADH (MW 709.4, reduced form) (Sigma-Aldrich, catalog number: N8129 )
    
11. 10x LDH-buffer (see Recipes)
    
12. 1x LDH-buffer (see Recipes)
    
13. LDH-standard (see Recipes)
    
14. Na-pyruvate-solution (see Recipes)
    
15. -NADH-solution (see Recipes)
    

Equipment

1. Pipette
   
2. CO₂-incubator for cell cultures
   
3. Multi-pipette (Eppendorf, model: Multipette^® plus )
   
4. Spectrophotometer for 96 well plate that can measure 340 nm and kinetic measurement (e.g., Dynex Technologies, model: MRX Microplate Reader )
   

Procedure