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.||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.|Luciferase ATP+D-luciferin+O2 oxyluciferin+AMP+PPi+CO2+light|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.||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: 10ml|Substrate: 120ul|Cosubstrate 120ul|ATP Enzyme: 120ul|ADP Enzyme: 120ul|Storage conditions: store all reagents at -20°C. This product is shipped on dry ice. Shelf life: 6 months after receipt.|Precautions: reagents are for research use only. Normal precautions for laboratory reagents should be exercised while using the reagents. Please refer to Material Safety Data Sheet for detailed information.||Assay Procedure:|Assays can be carried out in a tube or in a 96-well plate. For consistency, it is recommended that the time between the three luminescence measurements be the same for all samples.|1. Sample Preparation. For suspension cells, transfer 10ul of the cultured cells (103-104) into a white opaque 96 well plate. Adherent cells: culture 103-104 cells in white opaque microplate. At the time of assay, remove the culture medium immediately before adding 100ul ATP Reagent (see below).|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 95ul Assay Buffer with 1ul Substrate, 1ul Cosubstrate and 1ul ATP Enzyme. Add 90ul ATP Reagent to each well and mix by tapping the plate. After 1 min, read luminescence (RLU A) on a luminometer.|3. ADP Assay. Prepare ADP Reagent: for each 96-well, mix 5ul dH2O with 1ul ADP Enzyme. Ten minutes after reading the luminescence for ATP (RLU A), read the luminescence of the samples again (RLU B). This measurement provides the background prior to measuring ADP (i.e. the residual ATP signal). Immediately following reading RLU B, add 5ul ADP Reagent to each well and mix by tapping the plate or pipetting up and down. After 1 min. read luminescence (RLU C) on a luminometer.|4. Calculation: of ADP/ATP Ratio. Subtract RLU B from RLU C, then divide by RLU A:|ADP/ATP Ratio =|RLU C–RLU B|RLU A||Results Interpretation:|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.|2. Test gives lower ATP levels with an increase in ADP levels in comparison to control cells=apoptosis.|3. Test gives markedly lower ATP levels with greatly increased ADP levels in comparison to control cells=necrosis.