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Details for Product No. ABIN411720

ApoSENSOR™ ADP/ATP Ratio Assay Kit

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Target Name (Antigen)
Application
Biochemical Assay (BCA)
Pubmed 3 references available
Catalog no. ABIN411720
Quantity 200 tests
Price
489.50 $   Plus shipping costs $45.00
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Availability Backorder
Brand ApoSENSOR™
Sample Type Cell and Tissue Culture Lysate, Urine, Plasma, Serum, Biological Fluids
Specificity 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).
Components Nucleotide Releasing Buffer, ATP Monitoring Enzyme, ADP Converting Enzyme, Enzyme Reconstitution Buffer
Background 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.
Protocol A. Reagent Reconstitution and General Consideration: Reconstitute ATP Monitoring Enzyme and ADP Converting Enzyme each with 220 µL 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 µL per assay) for the number of assays to be performed in each experiment and freeze immediately at -70 °C for future use. The reconstituted enzymes are stable for up to 3 months at -70 °C after reconstitution. For more accurate handling, the enzyme can be dilute 10 fold with Nucleatide Releasing Buffer just before use, then use 10 µL 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 °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 µL/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 µL of the cultured cells (10 3 - 10 4 ) into luminometer plate. Add 100 µL of the Nucleotide Releasing Buffer. For adherent cells, remove culture medium and treat cells (10 3 - 10 4 ) with 100 µL of Nucleotide Releasing Buffer for 5 minutes at room temperature with gentle shaking.
3. To measure the ATP levels in the cells, add 1 µL 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 µL of ADP Converting Enzyme. Read the samples again in 1 minute in a luminometer (Data C). Note: The reconstituted Enzymes will be milky, cloudy solution, not clear solution). Aliquot enough enzymes (1 µL per assay) for the number of assays to be performed in each experiment and freeze immediately at -70 °C for future use. The reconstituted enzymes are stable for up to 3 months at -70 °C after reconstitution. For more accurate handling, the enzyme can be dilute 10 fold with Nucleatide Releasing Buffer just before use, then use 10 µL of the enzymes each for each assay.
The ApoSENSOR™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 °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 µL/well culture volume is recommended). Assay Protocol:
Restrictions For Research Use only
Storage -80 °C
Expiry Date 12 months
Product cited in: 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).

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).

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