Materials and Reagents

96 well plate, flat bottom (SARSTEDT, catalog number: 82.1581 )
Pipette tips
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.
Triton X-100 solution (Sigma-Aldrich, catalog number: 93443 )
Potassium phosphate monobasic (KH₂PO₄) (MW 136.1) (Sigma-Aldrich, catalog number: P5655 )
Dibasic potassium phosphate (K₂HPO₄; MW 174.2) or potassium phosphate trihydrate (K₂HPO₄·3H₂O; MW 228.2) (Sigma-Aldrich catalog number: P5504 )
Distilled water
Na-pyruvate (MW 110) (Sigma-Aldrich, catalog number: P2256 )
LDH standard (TruCal U) (DiaDys Diagnostic Systems, catalog number: 5 9100 99 10 063 )
-NADH (MW 709.4, reduced form) (Sigma-Aldrich, catalog number: N8129 )
10x LDH-buffer (see Recipes)
1x LDH-buffer (see Recipes)
LDH-standard (see Recipes)
Na-pyruvate-solution (see Recipes)
-NADH-solution (see Recipes)

Equipment

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

Procedure

Preparation of solutions (see Recipes)
Measurement

Warm up NADH and an appropriate aliquot of Na-pyruvate-solution (for one 96 well plate you need about 2.5 ml Na-pyruvate-solution) to room temperature or max. 37 °C, thaw appropriate amount of LDH-standard aliquots.
Pipette 50 µl cell culture medium (probe) in each well. A duplicate measurement of each cell culture well is recommended.
Pipette 25 µl LDH standard in 2 wells per plate. It is very important that you perform a standard measurement on each 96 well plate.
Add 200 µl NADH solution to each well and remove bubbles if necessary. The final concentration of NADH in the assay is 153.8 µM.
Add 25 µl Na-pyruvate solution to each well. The enzymatic reaction starts promptly. The final concentration of Na-pyruvate in the assay is 2.063 mM. Do not mix samples with pipets. Mixing should be initiated by using automated shaking by the plate reader before and in between of each measurement cycle. The reaction is within the linear range, if excess NADH is available for the enzymatic reaction. This lasts about 7-10 min. Ideally, pipetting of pyruvate and measurement of all cycles should be accomplished within this period. Do not start the reaction in more than one plate at once. It might be necessary to dilute the sample as specified in Figure 3.

Figure 3. Representative graph of a measurement. The blue line indicates an undiluted and the red line a diluted (F1:4, red) kinematic curve. Note that the undiluted sample shows a non-linear decrease in absorption at 340 nm.
Immediately start measurement of absorption with following parameters:

Wavelength: 340 nm.
Measurement cycles: 10.
Cycle time: 30 sec.
Shaking time: 5 sec.
Temperature: 37 °C (see also Notes).

Total lysis

Add Triton X-100 to the rest of cell culture medium. The final concentration should be 0.5%.
Incubate on 37 °C for 20 min. It is possible to incubate a longer time until to 4-6 h or overnight. This is not critical as long as you treat all groups equally.
Repeat steps 2b-2f from paragraph 2 (Measurement).

Data analysis

Calculate the decrease of absorption for each well in mAbs/min. In our hands the value is about 10-50 mAbs/min.
Note: Check, if the decrease of absorption is linear over the whole time. Otherwise, reduce the volume of probe or dilute them.
The decrease of absorption in LDH standard equates an LDH activity about 500 U/l. The LDH activity in standard is a little bit different in different batches. Calculate according to manufactures specifications.
Calculate the LDH activity for your probes using a rule of proportion.
Example:
1. Decrease of absorption in probe: 15 mAbs/min.
2. Decrease of absorption in LDH standard: 43 mAbs/min.
3. LDH activity in standard 466 U/l.
Equation: (15 mAbs/min x 466 U/l/43 U/l) = 162.5 U/l
Adjust the LDH activity for probe on the relation of probe volume and LDH standard volume.
Finally, calculate LDH activity as a percentage of LDH activity in the same cell culture sample after assessing LDH activity after total LDH release (‘full kill’).

Notes

The reaction rate is dependent on the temperature. In case of high LDH activity in your probe and an additional high temperature, an exhaustion of -NADH is possible and leads to a non-linear decrease of absorption.
Assessment of total LDH release after cell lysis (‘full kill’) is recommended for each cell culture well. There are some treatments, which lead to an increase of protein synthesis in cells, so that the amount of total LDH in the cells is increased as well. Normalization helps for better correlation as an indirect measure of cell death in such cases.
It is possible to store samples of supernatants at 4 °C if samples are sealed to avoid evaporation for two to three days with a minimal loss of LDH activity. Do not freeze samples.

Recipes

10x LDH-buffer
45.3 g KH₂PO₄
116.1 g K₂HPO₄ or 152 g K₂HPO₄·3H₂O
Dissolve in about 800 ml distilled water, adjust pH to 7.4 and fill up to 1,000 ml with distilled water
Store at 4 °C, The solution is stable for 12 month
1x LDH-buffer
Dilute 10x LDH buffer with distilled water 1:10
Store at 4 °C, long-term storage up to 12 months is possible
LDH-standard
Dissolve lysate of enzyme according to manufacture's protocol, prepare aliquots of 105 µl (that’s for measurement of two plates) and store at -20 °C. Avoid freeze and thaw cycles
Na-pyruvate-solution
Dissolve 1.25 g Na-pyruvate in 500 ml 1x LDH-buffer, results in 22.7 mM Na-pyruvate-solution
Store at 4 °C. The solution is stable for 18 month
-NADH-solution
Dissolve 3 mg -NADH in 20 ml 1x LDH-buffer, results in 211.4 µM solution
Note: 20 ml is the amount for one 96 well plate. This solution is stable only for 2 days at 4 °C.

Acknowledgments

This work was supported by the German Research Foundation (SFB TRR43 and HA5741/1-2 to Christoph Harms), the Federal Ministry of Education and Research (01 EO 08 01) for funding of the Center for Stroke Research Berlin (project ‘SUMO and stroke’ to Christoph Harms), and Berlin Institute of Health, TRG7, TP1 to Christoph Harms.

References

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