Plate luminometer

Example 40

Two fold serial dilutions of NADH, NADPH, NAD, and NADP (Sigma) were made in PBS starting from 0.313 uM. 10 ul of each dilution was transferred into wells of a 384-well plate. Detection reagents were made by adding 10 U/ml of rat diaphorase (Sigma), 40 uM proluciferin substrate PBI 4312 and NADP or NAD dependent enzyme amplification systems consisting of Glucose-6Phosphate Dehydrogenase (5 U/ml) and glucose-6P (0.5 mM) for NADP or Lactate Dehydrogenase (5 U/ml) and Lactate (40 mM) for NAD into Luciferin Detection Reagent (LDR; Promega Cat. No V8920). 10 ul of appropriate detection reagent was added to the dinucleotide samples. The reactions were incubated for 30 minutes at room temperature, and luminescence measured on a Tecan plate luminometer.

The results show that when the detection method described herein is combined with the dinucleotide specific amplification enzyme system, light is generated only in the samples containing appropriate dinucleotide (FIG. 31).

Example 39

Two fold serial dilutions of NADP (Sigma) were made in PBS starting from 0.5 μM. 25 μl of each dilution was transferred into wells of a 384-well plates. Detection reagent was made by adding 10 U/ml rat diaphorase (Sigma), 40 μM proluciferin substrate PBI 4312 and NADP dependent enzyme amplification system consisting of 0.5 U/ml glucose 6 phosphate Dehydrogenase (Sigma) and 500 μM glucose 6 phosphate (Sigma) into Luciferin Detection Reagent (LDR; Promega Cat. No V8920). 25μ of detection reagent was added to the NADP samples. The reaction was incubated for 30 minutes at room temperature, and luminescence measured using a Tecan plate luminometer.

The following example demonstrates the use of the pro-luciferin substrate PBI 4312 to detect and measure NADP Luminescence generated is indicative of the presence of NADP with the light output directly proportional to the amount of NADP present in the sample. The results show that the diaphorase and glucose 6 phosphate Dehydrogenase enzymes remain active in detection reagent, and all three enzymatic reactions can occur simultaneously (FIG. 30B).

Example 45

HepG2 cells were plated into wells of a 384-well plate at 5,000 cells/well in 25 ul of RPMI media containing 22 mM glucose or 10 mM galactose as an energy source. The cells were treated with 1 uM mitochondrial toxin antimycin or rotenone. At 4 and 24 hours after drug treatment, 25 μl of appropriate detection reagent was added to the cells. The NADH/NADPH detection reagent contained 10 U/ml rat diaphorase, 32 uM proluciferin substrate PBI 4312, 0.5 U/ml Glucose-6-Phosphate Dehydrogenase and 0.4 mM glucose-6-phosphate into Luciferin Detection Reagent. The NAD/NADH detection reagent contained 10 U/ml of rat diaphorase, 32 uM proluciferin substrate PBI 4312, 10 U/ml Lactate Dehydrogenase and 40 mM Lactate in Luciferin Detection Reagent. The reactions were incubated for 30 minutes at room temperature, and luminescence measured using a Tecan plate luminometer. The data are shown as % of dinucleotides remaining in the cells after treatment compared to untreated cells.

The results show that drug induced changes in cellular NAD(P)/NAD(P)H levels can be measured using the method described herein (FIG. 36).

Example 41

Two fold serial dilutions of PC3 cells were made in F12K media with 10% FBS. 25 ul of each dilution was transferred into wells of a 384-well plate. Detection reagent was made by adding 10 U/ml rat diaphorase (Sigma) and 40 μM proluciferin substrate PBI 4312 into Luciferin Detection Reagent (LDR; Promega Cat. No V8920). 25 μl of detection reagent was added directly to the cells. The reactions were incubated for 30 minutes at room temperature, and luminescence measured using a Tecan plate luminometer.

FIG. 32 shows the correlation between cell number and luminescence indicating a direct relationship between luminescence generated via proluciferin conversion and the total amount of reduced dinucleotides NADH/NADPH present in the cells. The results also show that detection system comprised of diaphorase and pro-luciferin PBI 4312 combined with Luciferase Detection Reagent (LDR) can be added directly to the cells to measure the amount of reduced dinucleotides present in the cells.

Example 38

Two fold serial dilutions of NAD (Sigma) were made in PBS starting from 0.25 μM. 25 μl of each dilution was transferred into wells of a 384-well plates. Detection reagent was made by adding 10 U/ml rat diaphorase (Sigma), 40 μM proluciferin substrate PBI 4312 and NAD dependent enzyme amplification system consisting of 5 U/ml lactate Dehydrogenase (Calbiochem) and 40 mM lactate (Sigma) into Luciferin Detection Reagent (LDR; Promega Cat. No V8920). 25 μl of the detection reagent was added to the NAD samples. The reactions were incubated for 30 minutes at room temperature, and luminescence measured using a Tecan plate luminometer.

The following example demonstrates the use of the pro-luciferin substrate PBI-4312 to detect and measure NAD Luminescence generated is indicative of the presence of NAD with the light output directly proportional to the amount of NAD present in the sample. The results show that the diaphorase and lactate dehydrogenase enzymes remain active in the detection reagent, and all three enzymatic reactions can occur simultaneously (FIG. 30A)