119107 ATP Assay Kit

119107
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Overview

Replacement Information

Key Specifications Table

Detection Methods
Luminescence

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      Description
      OverviewThe assay is based on the firefly luciferase-catalyzed oxidation of D-luciferin in the presence of ATP and oxygen, whereby the amount of ATP is quantified by the amount of light (hv) produced.
      Catalogue Number119107
      Brand Family Calbiochem®
      SynonymsLuciferin-Related Kit
      References
      Product Information
      Detection methodLuminescence
      Form200 Tests
      Format96-well, luminometer cuvette , or Beta counter
      Kit containsOne vial of Luciferase ATP Monitoring Enzyme, Enzyme Reconstitution Buffer, 1 bottle of Nucleotide Releasing Reagent, one vial of ATP Standard, and a user protocol.
      Quality LevelMQ100
      Applications
      Biological Information
      Assay time1 h
      Sample TypeBlood, milk, urine, soil, and sludge
      Physicochemical Information
      Dimensions
      Materials Information
      Toxicological Information
      Safety Information according to GHS
      Safety Information
      R PhraseR: 22;

      Product Usage Statements
      Intended useThe assay can be fully automatic for high throughput (10 s/sample) and is extremely sensitive (detects 10-100 mammalian cells/well). The high sensitivity of this assay has led to many other applications for detecting ATP production in various enzymatic reactions, as well as for detecting low level bacterial contamination in samples such as blood, milk, urine, soil, and sludge.
      Storage and Shipping Information
      Ship Code Dry Ice Only
      Toxicity Multiple Toxicity Values, refer to MSDS
      Storage ≤ -70°C
      Storage ConditionsUpon arrival store the entire contents of the kit at -70°C.
      Avoid freeze/thaw Avoid freeze/thaw
      Do not freeze Ok to freeze
      Packaging Information
      Transport Information
      Supplemental Information
      Kit containsOne vial of Luciferase ATP Monitoring Enzyme, Enzyme Reconstitution Buffer, 1 bottle of Nucleotide Releasing Reagent, one vial of ATP Standard, and a user protocol.
      Specifications

      Documentation

      ATP Assay Kit SDS

      Title

      Safety Data Sheet (SDS) 

      ATP Assay Kit Certificates of Analysis

      TitleLot Number
      119107
      User Protocol

      Revision28-July-2016 JSW
      SynonymsLuciferin-Related Kit
      Form200 Tests
      Format96-well, luminometer cuvette , or Beta counter
      Detection methodLuminescence
      StorageUpon arrival store the entire contents of the kit at -70°C.
      Intended useThe assay can be fully automatic for high throughput (10 s/sample) and is extremely sensitive (detects 10-100 mammalian cells/well). The high sensitivity of this assay has led to many other applications for detecting ATP production in various enzymatic reactions, as well as for detecting low level bacterial contamination in samples such as blood, milk, urine, soil, and sludge.
      BackgroundCell death (especially apoptosis) is an energy-dependent process that requires ATP. As ATP levels fall to a point where the cell can no longer perform basic metabolic functions, the cell will die. A typical apoptotic cell exhibits a significant decrease in ATP level. Therefore, loss of ATP level in the cell has been used as an indicator of cell death. In contrast, increased levels of ATP has been an indicator of cell proliferation. Calbiochem® brand ATP Assay Kit utilizes bioluminescent detection of the ATP levels for a rapid screening of apoptosis and cell proliferation simultaneously in mammalian cells. The assay utilizes luciferase to catalyze the formation of light from ATP and luciferin, and the light can be measured using a luminometer or Beta Counter.

      Figure 1: Calculations

      Materials provided• Nucleotide Releasing Buffer (Kit Component No. KP31201): used as a lysis buffer and reaction buffer
      • ATP Monitoring Enzyme (Kit Component No. KP31202)
      • Enzyme Reconstitution Buffer (Kit Component No. KP31203)
      • ATP, M.W. 551 (Kit Component No. KP31204)
      Precautions and recommendations Protect the ATP Monitoring Enzyme from light as much as possible.
      Keep ATP Monitoring Enzyme on ice during the assay.
      Ensure that the Nucleotide Releasing Buffer is at room temperature before use. The optimal temperature is 22°C.
      The ATP Assay kit significantly more sensitive than other methods used for cell viability assays. This method can detect as few as 10 cells, but as a general guide, we recommend using 103-104 cells per assay.
      Due to the high sensitivity of the ATP assay, avoid contamination with ATP from exogenous biological sources, such as bacteria or fingerprints.
      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).
      Reagent preparationReagent Reconstitution 1. Reconstitute ATP Monitoring Enzyme with 2ml of the Enzyme Reconstitution Buffer. Mix well by gentle pipetting. The reconstituted enzyme is stable for up to 2 months at 4°C. 2. Prepare an ATP standard solution by dissolving the 1 mg ATP into 1 ml of H2O. The solution is stable for several weeks at -20°C.
      Detailed protocol1. 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 (containing 103-104 cells) into luminometer plate. Add 100 µl of the Nucleotide Releasing Buffer. For adherent cells, remove culture medium and treat cells (103-104) with 100 µl of Nucleotide Releasing Buffer for 5 min at room temperature with gentle shaking.
      3. Add 10 µl ATP Monitoring Enzyme into the cell lysate. Read the sample within 1-2 min in a luminometer.
      4. Fold-decrease (or increase in the case of cell proliferation) in ATP levels can be determined by comparing these results with the levels of uninduced control.

      Note: The assay can be analyzed using cuvette-based luminometers or beta counters. When a beta counter is used it should be programmed in the "out of coincidence" (or luminescence mode) for measurement. The entire assay can also be carried out directly in a 96-well plate. It can also be programmed automatically using instrumentation with injectors. When using an injector 1 µl ATP Monitoring Enzyme can be diluted with 49 µl Nucleodtide Releasing Buffer.
      Standard curve

      Figure 2: Standard Curve

      If the absolute ATP amount in samples needs to be calculated, an ATP standard curve should be generated (using the ATP standard provided in the kit) together with the above assays. Add 10 µl of a series of dilutions of ATP (e.g., 1 mg/ml, 0.1 mg/ml, 0.01 mg/ml, 0.001 mg/ml, etc. Also includes a 0 mg/ml sample to measure background luminescence) to luminometer plates, then add 100 µl of Nucleotide Releasing Reagent and 1 µl of ATP Monitoring Enzyme. Read the samples in 1 min in a luminometer (as described above). The background luminescence should be subtracted from all readings. The amount of ATP in uninduced and induced experimental samples can then be calculated from the standard curve.

      Registered TrademarksCalbiochem® is a registered trademark of EMD Biosciences, Inc.
      Interactive Pathways™ is a trademark of EMD Biosciences, Inc.