2
Procedure
All samples and standards should be run in duplicate.
pNA Standards for Colorimetric Detection
Add 0, 4, 8, 12, 16, and 20 L of the 2 mM standard
solution into a 96
vacuum source. Place vial tray on system base with
tray numbers facing forward.
2. Place one to eight uncapped 12 × 32 mm vials in the
vial tray and place lid over vials.
3. Open doors directly
Solution into a 96 well plate, generating
0 (blank), 8, 16, 24, 32, and 40 nmole/well standards.
Add SDH Assay Buffer to each well to bring the volume
to 100 L.
Sample Preparation
Tissue samples
in the 96-well
MultiScreen Caco-2 system yields a better
dynamic range (MultiScreen Papp
0.5 – 26.3 vs. company B 96-well plate
0.4 – 17.5 x10-6 cm/sec).
Papp (x106 cm/sec) MultiScreen Caco-2 Company
to measure the total
-
32
P-ATP counts introduced into the reaction. Spot
5 l of the -
32
P-ATP Assay Cocktail on a precut
P81 strip. Dry the sample for 2 minutes and read
Buffer to prepare a 2 mM
(2 nmole/µL) GSH Standard Solution. Add 0, 4, 8, 12,
16, and 20 µL of the 2 mM GSH Standard Solution into
a 96 well plate to generate 0 (blank), 8, 16, 24, 32
Use soluble fraction for assay. Serum samples
may be assayed directly. Add 2–50 L samples into
duplicate wells of a 96 well plate. Bring samples to a
final volume of 50 L with LDH Assay Buffer
duplicate.
NADH Standards for Colorimetric Detection
Add 0, 2, 4, 6, 8, and 10 µL of the 1.25 mM NADH
standard solution in duplicate into a 96 well plate,
generating 0 (blank), 2.5, 5, 7.5, 10, and
12.5
Buffer to prepare a 1 mM
standard solution. Add 0, 2, 4, 6, 8, and 10 µL of the
1 mM standard solution into a 96 well plate, generating
0 (blank), 2, 4, 6, 8, and 10 nmole/well standards. Add
ADH
the Lipase Assay Buffer to prepare a
10 µM standard solution. Add 0, 2, 4, 6, 8, and 10 µL of
the 10 µM standard solution into a 96 well plate,
generating 0 (blank), 20, 40, 60, 80, and 100