ApoSENSOR™ ADP/ATP Ratio Assay Kit

Application: Biochemical Assay (BCA)

Catalog no.: ABIN411720

Additional Information

Sample Type: Cell and Tissue Culture Lysate, Urine, Plasma, Serum, Biological Fluids

Components: Nucleotide Releasing Buffer , ATP Monitoring Enzyme , ADP Converting Enzyme , Enzyme Reconstitution Buffer

Description: The changes in ADP/ATP ratio have been used to differentiate the different modes of cell death and viability. Increased levels of ATP and decreased levels of ADP have been recognized in proliferating cells. In contrast, decreased levels of ATP and increased levels of ADP are recognized in apoptotic cells. The decrease in ATP and increase in ADP are much more pronounced in necrosis than apoptosis. The ApoSENSOR TM ADP/ATP Ratio Assay kit utilizes bioluminescent detection of the ADP and ATP levels for a rapid screening of apoptosis, necrosis, growth arrest, and cell proliferation simultaneously in mammalian cells. The assay utilizes the enzyme luciferase to catalyze the formation of light from ATP and luciferin, and the light can be measured using a luminometer or Beta Counter. ADP level is measured by its conversion to ATP that is subsequently detected using the same reaction. The assay can be fully automatic for high throughput and is highly sensitive (detects 100 mammalian cells/well).

Application Details

Protocol: A. Reagent Reconstitution and General Consideration: Reconstitute ATP Monitoring Enzyme and ADP Converting Enzyme each with 220 μ l of the Enzyme Reconstitution Buffer. Mix gently by inversion (Note: The reconstituted Enzymes will be milky, cloudy solution, not clear solution). Aliquot enough enzymes (1 μ l per assay) for the number of assays to be performed in each experiment and freeze immediately at –70 o C for future use. The reconstituted enzymes are stable for up to 3 months at –70 o C after reconstitution. For more accurate handling, the enzyme can be dilute 10 fold with Nucleatide Releasing Buffer just before use, then use 10 μ l of the enzymes each for each assay. The ApoSENSOR TM kit is significantly more sensitive than other methods used for cell viability assays. The method can detect as few as 10 cells, but as a general guide, we recommend using 1 x 10 4 cells per assay. Avoid contamination with ATP from exogeneous biological sources, such as bacteria or fingerprints. Ensure that the Nucleotide Releasing Buffer is at room temperature before use. The optimal temperature is 22 o C. Keep other enzymes on ice during the assay and protect from light as much as possible. The assay can be performed using either a single tube or a white walled 96-well luminometer plate (100 μ l/well culture volume is recommended). 1. Induce apoptosis in cells by desired method. Concurrently incubate a control culture without induction. 2. For suspension cells, transfer 10 μ l of the cultured cells (10 3 – 10 4 ) into luminometer plate. Add 100 μ l of the Nucleotide Releasing Buffer. For adherent cells, remove culture medium and treat cells (10 3 – 10 4 ) with 100 μ l of Nucleotide Releasing Buffer for 5 minutes at room temperature with gentle shaking. 3. To measure the ATP levels in the cells, add 1 μ l of the ATP Monitoring Enzyme into the cell lysate. Read the sample in 1 minute in a luminometer (Data A). 4. To measure ADP levels in the cells, read the samples (from step 3) in 10 minutes (Data B), then add 1 μ l of ADP Converting Enzyme. Read the samples again in 1 minute in a luminometer (Data C).

Application Notes: Note: The reconstituted Enzymes will be milky, cloudy solution, not clear solution). Aliquot enough enzymes (1 μ l per assay) for the number of assays to be performed in each experiment and freeze immediately at –70 o C for future use. The reconstituted enzymes are stable for up to 3 months at –70 o C after reconstitution. For more accurate handling, the enzyme can be dilute 10 fold with Nucleatide Releasing Buffer just before use, then use 10 μ l of the enzymes each for each assay. • The ApoSENSOR TM kit is significantly more sensitive than other methods used for cell viability assays. The method can detect as few as 10 cells, but as a general guide, we recommend using 1 x 10 4 cells per assay. Avoid contamination with ATP from exogeneous biological sources, such as bacteria or fingerprints. • Ensure that the Nucleotide Releasing Buffer is at room temperature before use. The optimal temperature is 22 o C. Keep other enzymes on ice during the assay and protect from light as much as possible. • The assay can be performed using either a single tube or a white walled 96-well luminometer plate (100 μ l/well culture volume is recommended). Assay Protocol:

Storage: -70°C

Expiry Date: 12 months

Restrictions: For Research Use only

Publications

Drukala, Urbanska, Wilk et al.: "ROS accumulation and IGF-IR inhibition contribute to fenofibrate/PPARalpha -mediated inhibition of Glioma cell motility in vitro." in: Molecular cancer, Vol. 9, pp. 159, 2010 (PubMed).

Product: Tokmakov, Iguchi, Iwasaki et al.: "Unfertilized frog eggs die by apoptosis following meiotic exit." in: BMC cell biology, Vol. 12, Issue 1, pp. 56, 2011 (PubMed).

Heo, Park, Kim et al.: "DJ-1 Null Dopaminergic Neuronal Cells Exhibit Defects in Mitochondrial Function and Structure: Involvement of Mitochondrial Complex I Assembly." in: PLoS ONE, Vol. 7, Issue 3, pp. e32629, 2012 (PubMed).