Changes in the ADP/ATP ratio have been used to differentiate modes of cell death and viability. Increased levels of ATP and decreased levels of ADP signify proliferating cells. Conversely, decreased levels of ATP and increased levels of ADP represent apoptotic or necrotic cells where the decrease in ATP and increase in ADP is much more pronounced in necrosis versus apoptosis. BioAssay Systems’ EnzyLightTM ADP/ATP Ratio Assay Kit provides a  rapid method to measure ADP and ATP levels for the screening of apoptosis, necrosis and cell proliferation in mammalian cells. The assay involves two steps. In the first step, the working reagent lyses cells to release ATP and ADP. In the presence of luciferase, ATP immediately reacts with the Substrate D-luciferin to produce light. The light intensity is a direct measure of intracellular ATP concentration.

In the second step, the ADP is converted to ATP through an enzyme reaction. This newly formed ATP then reacts with the D-luciferin as in the first step. Due to a special formulation of the reagent system which greatly stabilizes the light signal generated by the luciferase reaction, the luminescence from the initial ATP measurement remains stable throughout this assay. Therefore, the second light intensity measured represents the total ADP and ATP concentration in the sample. This non-radioactive, homogeneous cell-based assay is performed in microplates. The reagent is compatible with all culture media and with all liquid handling systems for high-throughput screening applications in 96- well and 384-well plates.

KEY FEATURES

Safe. Non-radioactive assay.

Homogeneous and convenient. "Mix-incubate-measure" type assay. No wash and reagent transfer steps are involved.

Robust and amenable to HTS: Z’ factors of 0.5 and above are routinely observed in 96-well and 384-well plates. Can be readily automated on HTS liquid handling systems for processing thousands of samples per day.

APPLICATIONS

Apoptosis and Necrosis determination in cells.

Cell proliferation: effects of cytokines, growth factor, nutrients.

Drug discovery: high-throughput screening for anticancer drugs.

KIT CONTENTS

Assay Buffer: 10 mL

Substrate: 120 μL

Cosubstrate 120 μL

ATP Enzyme: 120 μL

ADP Enzyme: 120 μL well. Rock plate lightly to mix and incubate for desired period of time (e.g. overnight).

2. ATP Assay. Bring Assay Buffer, Substrate and Cosubstrate to room temperature. Thaw enzyme on ice or at 4°C. Fresh Reconstitution is recommended. Store unused reagents including the enzyme at -20°C. ATP Reagent. For each 96-well, mix 95 μL Assay Buffer with 1 μL Substrate, 1 μL Cosubstrate and 1 μL ATP Enzyme. For each 384-well, mix 30 μL Assay Buffer with 0.3 μL Substrate, 0.3 μL Cosubstrate and 0.3 μL ATP Enzyme. Add ATP Reagent to each well (90 μL/96well, 25 μL/384well) and mix by tapping the plate. Incubate for 10 minutes at room temperature. Read luminescence (RLU A) on a luminometer. For most luminometers (Berthold Luminometer, LJL Analyst, Top Count, MicroBeta Counters, CLIPR and LeadSeeker), integration time of 0.1 to 5 sec is appropriate.

3. ADP Assay. Prepare ADP Reagent: for each 96-well, mix 12 μL dH2O with 1 μL ADP Enzyme. For each 384-well, mix 3 μL dH2O with 0.25 μL ADP Enzyme. Add ADP Reagent to each well (10 μL/96well, 2.5 μL/384well) and mix by tapping the plate or pipetting up and down. Incubate for 10 minutes at room temperature. Read luminescence (RLU B) on a luminometer.

4. Calculation of ADP/ATP Ratio. Subtract RLU A from RLU B, then divide by Data A:

The interpretation of different ratios obtained may vary significantly according to the cell types and conditions used. However, the following may be used as guidelines:

1. Test gives markedly elevated ATP levels with no significant increase in ADP levels in comparison to control cells = proliferation. (Ratio <0.1)

2. Test gives lower ATP levels with an increase in ADP levels in comparison to control cells = apoptosis. (Ratio ~0.1-1.0)

3. Test gives markedly lower ATP levels with greatly increased ADP levels in comparison to control cells = necrosis. (Ratio > 1.0)

[1]. Bradbury DA, et al (2000). Measurement of the ADP:ATP ratio in human leukaemic cell lines can be used as an indicator of cell viability, necrosis and apoptosis. J Immunol Methods. 240:79-92.

[2]. Chen-Scarabelli C, et al (2004). Turning necrosis into apoptosis: the exacting task that can enhance survival. Am Heart J. 148(2):196-9.

[3]. Crouch S, et al (1993). The use of ATP Bioluminescence as a measure of cell proliferation and cytotoxicity. J Immunol Methods, 160(1): 81-8.