Table 3 Brief description of assessment of viability of brain slices based on the activity of released lactate dehydrogenase

1.

Preparation of Background absorbance control. Take 200 μl of aECF buffer from the scintillation vial containing the drug cassette before incubation and mix with 200 μl of blank aECF (store at +4°C pending analysis)

2.

Preparation of Low control. Take 200 μl of aECF buffer from the beaker containing the drug cassette five minutes after transferring the freshly prepared brain slices into the beaker and mix with 200 μl of blank aECF buffer (store at +4°C pending analysis)

3.

Preparation of Samples. Take 200 μl of aECF buffer from the beaker containing the drug cassette and brain slices after 5 hours of incubation and mix with 200 μl of blank aECF buffer (store at +4°C pending analysis)

4.

Preparation of High control. Place one (rat) or 3 (mouse) weighed brain slices in an Eppendorf tube after 5 hours of incubation with the drug cassette, and add 9 volumes (w/v) of 2% Triton X-100 solution in aECF. Put Eppendorf tubes in ultrasound bath for 1 hour at +4°C. Then incubate the tubes for 30 minutes in a water bath at 37°C. Centrifuge at 10000 rpm for 5 minutes at +4°C Take supernatant and store at +4°C pending analysis

5.

Preparation of the samples for analysis, see Table 4. Perform all tests in triplicate Protect the plate from light after addition of freshly prepared reaction mixture

6.

Incubate the plate for up to 25 minutes at room temperature

7.

Measure the absorbance of the samples at 492 nm (use 690 nm as a reference wavelength)