ADP/ATP ratio

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EnzyLightTM ADP/ATP Ratio Assay Kit (ELDT-100)

Bioluminescent Assay for ADP/ATP Ratio

DESCRIPTION

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:

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. (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)

LITERATURE

[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.

EnzyLightTM ATP Assay Kit (EATP-100)

Rapid bioluminescent determination of ATP

DESCRIPTION

Adenosine 5'-triphosphate (ATP) is the chemical energy for cellular metabolism and is often referred to as “energy currency" of the cell. ATP is produced only in living cells during photosynthesis and cellular respiration and consumed in cellular processes including biosynthetic reactions, motility and cell division. It is a key indicator of cellular activity and has been utilized as a measure of cell viability and cytotoxicity in research and drug discovery. BioAssay Systems’ EnzyLightTM ATP Assay Kit provides a rapid method to measure intracellular ATP. The single working reagent lyses cells to release ATP, which, in the presence of luciferase, immediately reacts with the Substrate D-luciferin to produce light. The light intensity is a direct measure of intracellular ATP concentration.

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.

Sensitive and accurate. As low as 0.1 μM ATP or 40 cells can be quantified.

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 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

ATP determination in cells and other biological samples.

KIT CONTENTS

Assay Buffer: 10 mL

Substrate: 120 μL

ATP Enzyme: 1 20 μL

Standard: 100 μL 3 mM ATP

Storage conditions: store all reagents at -20°C. This kit is shipped on dry ice. Shelf life of at least 6 months.

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

1. Standard Curve. Prepare 1000 μL 30 μM ATP Premix by mixing 10 μL 3 mM Standard and 990 μL distilled water (for cell culture samples dilute ATP in culture media). Dilute standard as follows. Transfer 100 μL standards into wells of a white opaque 96-well plate.

Samples. Add 100 μL sample per well in separate wells. For tissue samples, homogenize 20 mg sample in 200 μL of cold phosphate-buffered saline, spin at 12,000 g for 5 min to pellet any debris. Transfer 1-100 μL supernatant to each well and bring the volume to 100 μL with PBS. Test several doses of the sample and choose the readings that are within the standard curve range for ATP calculation. For cell cultures, plate cells (100 μL/96well plate, 25 μL/384well plate) in white opaque tissue culture plates. If desired, add 5 μL test compounds and controls dissolved in PBS or culture medium per well. Rock plate lightly to mix and incubate for desired period of time (e.g. overnight).

2. Assay. Bring Assay Buffer and Substrate to room temperature. Thaw enzyme on ice or at 4°C. Fresh Reconstitution is recommended. Store unused reagents including the enzyme at -20°C. For each 96-well, mix 95 μL Assay Buffer with 1 μL Substrate and 1 μL ATP Enzyme. Add 90 μL Reconstituted Reagent to each well. For each 384-well, mix 30 μL Assay Buffer with 0.3 μL Substrate and 0.3 μL ATP Enzyme. Add 25 μL Reconstituted Reagent to each well. Mix by tapping the plate. Incubate for 10 minutes at room temperature. 3. Read luminescence 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.

GENERAL CONSIDERATIONS

Signal stability. After adding the Reconstituted Reagent, the luminescence signal is stable for about 20 min and decreases slow thereafter.

LITERATURE

[1]. Kangas L, et al. (1984). Bioluminescence of cellular ATP: a new method for evaluating agents in vitro. Medical Biology, 62: 338 - 343.

[2]. Zhelev Z, et al (2004). Phenothiazines suppress proliferation and induce apoptosis in cultured leukemic cells without any influence on the viability of normal lymphocytes. Phenothiazines and leukemia. Cancer Chemother Pharmacol. 53(3):267-75.

[3]. Ingram PR, et al (2004). A comparison of the effects of ocular preservatives on mammalian and microbial ATP and glutathione levels. Free Radic Res.

QuantiChromTM ATPase/GTPase Assay Kit (DATG-200)

DESCRIPTION

ATPases and GTPases catalyze the decomposition of ATP or GTP into ADP or GDP and free phosphate ion. These enzymes play key roles in transport, signal transduction, protein biosynthesis and cell differentiation. BioAssay Systems’ QuantiChromTM ATPase/GTPase Assay Kit offers a highly sensitive method for determining ATPase/GTPase activities in a microplate format. Its proprietary formulation features a single reagent for accurate determination of enzyme activity in 30 min at room temperature. The improved malachite green reagent forms a stable dark green color with liberated phosphate, which is measured on a plate reader (600 - 660 nm).

KEY FEATURES

High sensitivity: detection of as little of 2 pmoles of free phosphate.

Fast and convenient: single reagent, homogeneous “mix-and-measure” assay allows quantitation of enzyme activity within 30 minutes.

Robust and amenable to HTS: detection at 620nm greatly reduces potential interference by colored compounds. Z’ factors of >0.7 are observed in 96-well and 384-well plates. Can be readily automated on HTS liquid handling systems.

APPLICATIONS

Determination of ATPase and GTPase activity.

Drug Discovery: high-throughput screen for ATPase or GTPase inhibitors.

KIT CONTENTS: 200 ASSAYS IN 96-WELL PLATE

Reagent: 50 mL Assay Buffer: 10 mL

Standard: 1mL 1 mM phosphate

Storage conditions. The reagents and standard are stable for one year when stored at 4°C.

Precautions: reagent contains 0.27 M H2SO4. Normal precautions for laboratory reagents should be exercised while using the reagents. Please refer to Material Safety Data Sheet for detailed information.

Important: All reagents must be brought to room temperature before use. Before each assay, it is important to check that enzyme preparations and assay buffers do not contain free phosphate. This can be conveniently done by adding 200 μL of the Reagent to 40 μL sample solution. The blank OD values at 620 nm should be lower than 0.3. If the OD readings are higher than 0.3, check phosphate level. Lab detergents may contain high levels of phosphate. Make sure that lab wares are free from contaminating phosphate after thorough washes.

ACTIVITY DETERMINATION IN 96-WELL PLATE

1. Preparation of phosphate standards. Prepare 800 μL Premix solution containing 50 μM phosphate by mixing 40 μL 1 mM phosphate standard with 760 μL distilled water. Number the tubes. Dilute standards as shown in the following Table. Pipette 40 μL standard in duplicate into wells of a clear-bottom 96-well plate.

2. Perform a series dilution of enzyme in assay buffer. Set up 40-μL reactions and a control with no enzyme in separate wells. Incubate the reaction for desired period of time (e.g. 30 min).

3. Add 200 μL Reagent and incubate 30 min at room temperature. Please note: use of a multi-chanel pipettor is recommended. The Reagent terminates the enzyme reaction and generates color with the free phosphate produced in the enzyme reaction.

4. Read OD620nm on a plate reader.

5. Enzyme activity. Calculate DOD values by subtracting OD values in reaction and control wells. Choose an enzyme concentration that gives a DOD of 0.5 to 1, this will ensure that substrate hydrolysis (<10%) is within the linear kinetics of reaction. Compute the concentration of free phosphate produced from the standard curve. 1 Unit of activity is the amount of enzyme that catalyzes the production of 1 μmole of free phosphate per minute under the assay conditions.

INHIBITOR ASSAY IN 96-WELL PLATE

To evaluate an inhibitor or perform HTS, use the optimal enzyme concentration determined above. Incubate enzyme and inhibitor first for a certain period of time, before adding the substrate. At the end of reaction, add 200 μL Reagent for phosphate determination.

ASSAYS IN 384-WELL PLATE

The procedure is similar as in the 96-well plate assay, except that 20 μL standards or 20 μL reaction mixture (10 μL Assay Buffer, 5 μL 4 mM ATP, 5 μL enzyme) are mixed with 80 μL Reagent.

GENERAL CONSIDERATIONS

Materials. Use ultrapure ATP and GTP. The provided 2x assay buffer contains 40 mM Tris, 80 mM NaCl, 8 mM MgAc2, 1 mM EDTA, pH 7.5. Other buffers (Hepes, Mes, Mops) can be used. Assay is compatible with 1 mM DTT, 2mM b-mercaptoethanol, 0.5 mg/mL BSA and 5% DMSO.

Phosphate assays in 96-well plate. 1. H2O, 2. Phosphate, 3. ATP in H2O, 4. ATP/Phosphate in H2O, 5, 7, 9, 11, 13: ATP in Assay Buffer with, where indicated, 0.5 mg/mL BSA, 1 mM DTT, 2 mM b-mercaptoethanol (bME) and 5% DMSO. 6, 8, 10, 12, 14: ATP/Phosphate in Assay Buffer. Phosphate and ATP were at 50 μM and 1 mM, respectively. The assay is not affected by these components.