Store at -20°C

Cyclic AMP XP® Assay Kit 31 Kit n

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#4339

(96 assays)

rev. 01/21/16

For Research Use Only. Not For Use In Diagnostic Procedures.

HRP-linked cAMP

11 ml

Red

1 ml

Colorless

TMB Substrate

11 ml

Colorless

11 ml

Colorless

STOP Solution Sealing Tape

2 sheets

20X Wash Buffer

10 ml

Colorless

Cell Lysis Buffer (10X) #9803

15 ml

Yellowish

* 12 8-well modules -Each module is designed to break apart for 8 tests.

Note: This kit contains mixed storage components. Please store this entire kit at -20° C for long term storage. Upon first use, please allow components to thaw and then store each component as indicated on individual component labels.

4

110

0.5 mM IBMX No IBMX

0.5 mM IBMX No IBMX

90

3

70 2

1

50

EC50: 0.58 µM (No IBMX) 0.09 µM (0.5 mM IBMX)

30 10

0.0

3.0 2.5

-4

-3

-2

-1 0 1 Log [Forskolin] (µM)

2

0.0

3

-4

-3

-2

-1 0 1 Log [Forskolin] (µM)

2

3

Figure 2: Treatment of CHO cells with Forskolin #3828 increases cAMP concentration as detected by Cyclic AMP XP® Assay Kit #4339. CHO cells were seeded at 4x104 cells/well in a 96-well plate and incubated overnight. Cells were either left untreated or pretreated with 0.5 mM IBMX for 30 minutes prior to forskolin treatment (15 minutes) and lysed with 1X Cell Lysis Buffer #9803. The absorbance values (left) and percentage of activity (right) are shown above. The percentage of activity is calculated as follows: % activity=100X[(A-Abasal)/ (Amax-Abasal)], where A is the sample absorbance, Amax is the absorbance at maximum stimulation (i.e., high forskolin concentration), and Abasal is the absorbance at basal level (no forskolin). Forskolin directly activates adenylyl cyclases and increases cellular cAMP concentration. IBMX is a non-specific inhibitor of cAMP and cGMP phosphodiesterases and promotes accumulation of cAMP and cGMP in cells.

1.5 1.0 0.5 0.0 0.0

1

10

100

1000

Figure 1: cAMP Standard was diluted in 1X Cell Lysis Buffer #9803 and samples were assayed following the Cyclic AMP XP® Assay Kit protocol. This standard curve is for demonstration purposes only; users should generate a standard curve for each sample set in order to accurately determine cAMP concentration.

Absorbance 450 nm

[cAMP, nM]

3.0

110

2.5

90

2.0

70

1.5 1.0

50

0.5

10

0.0

-10 -3

-2

-1 0 Log [Isoproterenol] (µM)

1

EC50= 0.027 µM

30

-2

-1

0

1

Log [Isoproterenol] (µM)

Figure 3: Treatment of 293 cells with isoproterenol increases the cAMP concentration as detected by Cyclic AMP XP® Assay Kit #4339. 293 cells were seeded at 3x104 cells/well in a 96-well plate and incubated overnight. Cells were pretreated with 0.5 mM IBMX for 30 minutes prior to isoproterenol treatment (3 minutes) and lysed with 1X Cell Lysis Buffer #9803. The absorbance values (left) and percentage of activity (right) are shown above. The percentage of activity is calculated as follow: % activity=100X[(A-Abasal)/(Amax-Abas)], where A is the absorbance of the sample, Amax is the absorbance at maximum stimulation (i.e., high isoproterenol concentration), al and Abasal is the absorbance at basal level (no isoproterenol). Isoproterenol is a β-adrenoceptor agonist and activates b-2 adrenergic receptors (ADRB2) that are endogenously expressed on 293 cells. Activation of ADRB2 then leads to activation of adenylyl cyclase and synthesis of cAMP as its second messenger.

page 1 of 3

2.0

% Activity

Absorbance 450 nm

Solution

96 assays

cAMP Standard (2.4 µM)

3.5

© 2011 Cell Signaling Technology, Inc.

Kit Quantity

cAMP XP® Rabbit mAb Coated Microwells

% Activity

Specificity/Sensitivity: The immunoreactivity of this kit was tested against the following: ADP, AMP, ATP, cAMP, cGMP, cIMP, cTMP, CTP, GDP, GMP and GTP. Relatively minor cross-reactivity was observed with cGMP and cIMP, with 10 fold higher sensitivity for cAMP compared to either cGMP or cIMP. No cross-reactivity was observed with any of the other factors tested. Kit sensitivity, as shown in Figure 1, demonstrates a dynamic range of 0.2 to 12 nM of cAMP. Changes in cellular cAMP levels following specific treatments are shown in Figure 2 (CHO cells) and Figure 3 (293 cells).

Products Included

Absorbance 450 nm

Description: The Cyclic AMP XP® Assay Kit is a competition enzyme-linked immunoassay used to determine cAMP levels in cells or tissues of interest. In this assay, cAMP found in test sample competes with a fixed amount of HRP-linked cAMP for binding to an anti-cAMP XPTM Rabbit mAb immobilized onto a 96-well plate. Following washing to remove excess sample cAMP and HRP-linked cAMP, HRP substrate TMB is added to develop color. Because of the competitive nature of this assay, the magnitude of the absorbance for this developed color is inversely proportional to the quantity of sample cAMP. Measurement of absorbance using the cAMP Standard allows calculating the absolute amount of cAMP in a sample of interest.

Background: Cyclic adenosine 3’,5’-monophosphate (cAMP) is an important second messenger involved in many signal transduction pathways in different cell types of numerous species (13). In mammalian cells this important molecule is produced by adenylyl cyclases (AC). Extracellular stimuli such as neurotransmitters, hormones, chemokines, lipid mediators and drugs, can modulate AC activity to increase or decrease cAMP production by binding to a large number of transmembrane G protein-coupled receptors (4). The degradation of cAMP to AMP is catalyzed by phosphodiesterases that are regulated by intracellular nucleotide concentrations, phosphorylation, or binding of Ca2+/calmodulin and other regulatory proteins (5). A set of diverse molecules, including cAMP-dependent protein kinase (PKA), cyclic nucleotide-gated ion channels, and exchange proteins that are activated by cAMP (Epac), mediate downstream cAMP signaling (6,7). cAMP modulates various biological processes including metabolism, differentiation, cardiac cell functions, neuronal signaling, cell adhesion, and immune functions (5-7). Background References: (1) Serezani, C.H. et al. (2008) Am J Respir Cell Mol Biol 39, 127-32. (2) Beavo, J.A. and Brunton, L.L. (2002) Nat Rev Mol Cell Biol 3, 710-8. (3) Kopperud, R. et al. (2003) FEBS Lett 546, 121-6. (4) Kamenetsky, M. et al. (2006) J Mol Biol 362, 623-39. (5) Cheng, J. and Grande, J.P. (2007) Exp Biol Med (Maywood) 232, 38-51. (6) Holz, G.G. et al. (2006) J Physiol 577, 5-15.

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page 2 of 3

© 2011 Cell Signaling Technology, Inc.

(7) Taylor, S.S. et al. (2008) Biochim Biophys Acta 1784, 16-26.

#4339

cAMP XP® Assay Kit Protocol C Assay

1. Bring all microwell strips to room temperature before use. 2. Prepare 1X Wash Buffer by diluting 20X Wash Buffer (included in each kit) in Milli-Q or equivalently purified water. 3. Dilute the 10X Cell Lysis Buffer #9803 to 1X in Milli-Q or equivalently purified water. 1 mM phenylmethylsulfonyl fluoride (PMSF) should be added fresh each time. This buffer can be stored at 4°C for short-term use (1–2 weeks).

B Cell Lysate Preparation

© 2011 Cell Signaling Technology, Inc.

1. Plate cells of interest in 96-well plate (typically between 6-100 X 103 cells/well) and incubate overnight under appropriate cell culture conditions. 2. Rinse cells with 200 μl warm PBS, then add test compounds in serum free mediums and incubate cells for the desired time period. 3. Rinse cells twice with 200 µl ice cold PBS, and then add 100 μl/well 1X lysis buffer, keep cells on ice for 5 to 10 minutes. Note: If cell debris is observed it can be removed by brief centrifugation of the plate and transfer of the clear lysates to a new 96 well plate.

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1. Bring all kit components to room temperature. 2. Make cAMP standard in the 1X Cell Lysis buffer: Take 50 μl of the cAMP standard (2.4 µM) and add it to 450 μl diluent to get 240 nM cAMP. Perform a 1:3 serial dilution of this standard to get 80 nM, 26.7 nM, 8.9 nM, 3.0 nM, 1.0 nM, 0.3 nM and 0nM. The diluent without cAMP will serve as the 0 nM cAMP.  Note: The standard curve is used to calculate the absolute amount of cAMP in the sample and is necessary for each assay. 3. Add 50 µl of the HRP-linked cAMP solution and 50 μl sample to the cAMP assay plate. Cover the plate and incubate at room temperature for 3 hours on a horizontal orbital plate shaker. 4. Discard plate contents and wash wells 4 times with 200 μl /well of 1X Wash Buffer. Make sure to discard all liquid after each wash but do not allow wells to completely dry. 5. Add 100 μl TMB substrate. 6. Incubate for 30 minutes at room temperature. Note: Watch the color as it being developed since it may be necessary to stop the reaction before 30 minutes. 7. Add 100 μl STOP solution. 8. Measure absorbance at 450 nm (for optimal results, read the plate within 30 minutes after adding STOP solution).

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page 3 of 3

A Reagent Preparation

#5456 MSDS

MSDS

Material Safety Data Sheet (MSDS) for cAMP Assay Kits Rev. 03/25/11

I. Identification:

VIII-XIII.

I. Identification:

Refer to individual MSDS for kit components for Sections 8-13 information: Exposure Controls/ Personal Protection, Physical and Chemical Properties, Stability and Reactivity, Toxicological Information, Ecological information, Disposal Considerations.

Product name: cAMP Assay Kit, Cyclic AMP XP™ Chemiluminescent Assay Kit Product Catalog: 4339, 8019 CAS number: None Manufacturer Supplier: Cell Signaling Technology 3 Trask Lane Danvers, MA 01923 USA 1-978-867-2300 TEL 1-978-867-2400 FAX 1-978-578-6737 Emergency Phone

D.O.T. Proper Shipping Name: None. This substance is considered non-hazardous for transport. IATA Proper Shipping Name: None. This substance is considered non-hazardous for air transport.

XV. Regulatory Information:

Preparation Name: cyclic AMP Assay Kit CAS#: None

III. Hazard Identification: This product is For Research Use Only. Please refer to the attached material safety data sheets for information specific to hazards associated with this product: • Cell Lysis Buffer (CST#9803) MSDS • TMB (CST#7004) • STOP Solution (CST#7002)

EU: Not classified. OSHA: Not listed. Canadian DSL: Not listed. SARA 302, 313: Not listed. SARA 311/312: Not listed. Massachusetts Right To Know: Not Listed. Pennsylvania Right To Know: Not Listed. New Jersey Right To Know: Not Listed. California Prop. 65: Not Listed.

IV. First Aid Measures for Hazardous Ingredient:

!! CAUTION: This product is not for use in humans. It is intended for research purposes only. To the best of our knowledge, the chemical, physical, and toxicological properties of this material have not been established. Emergency Overview : OSHA: No known hazards. This substance is not classified as dangerous according to Directive 67/548/EEC.

This product is not intended for use in humans. To the best of our knowledge, this document is accurate. It is intended to serve as a guide for safe use of this product in a laboratory setting by experienced personnel. The burden of safe use of this material rests entirely with the user. The above information is believed to be accurate but is not necessarily all-inclusive and shall be used only as a guide. Cell Signaling Technology, Inc., shall not be held liable for any damage resulting from the handling of or from contact with the above product.

IV. First Aid Measures for Hazardous Ingredient:

Inhalation: Remove to fresh air. If breathing is difficult, get medical attention. Ingestion: If person is conscious, wash out mouth with water. Get medical attention. Skin contact: In case of contact, immediately wash skin with soap and water for at least 15 minutes. Remove contaminated clothing. Wash clothing before reuse. Eye contact: In case of contact with eyes, immediately flush eyes water for at least 15 minutes. Get medical attention.

V. Fire Fighting Measures:

Flash Point: Not applicable. Autoignition Temperature: Not applicable. Explosion: Not applicable. Fire extinguishing media: Water spray, dry chemical, foam, or carbon dioxide. Firefighting: : Wear protective clothing and self-contained breathing apparatus to prevent contact with skin and eyes. Specific Hazard: None.

VI. Accidental Release Measures:

II. Composition/Information on Ingredients: Substance Name: Adenosine 3.5’-cyclic monophosphate-horseradish peroxidase conjugate Synonyms: cAMP-HRP conjugate CAS#: none

III. Hazard Identification:

!! CAUTION: This product is not for use in humans. It is intended for research purposes only. To the best of our knowledge, the chemical, physical, and toxicological properties of this material have not been established. Emergency Overview : OSHA: No known hazards. This substance is not classified as dangerous according to Directive 67/548/EEC.

IV. First Aid Measures for Hazardous Ingredient:

Inhalation: Remove to fresh air. If breathing is difficult, get medical attention. Ingestion: If person is conscious, wash out mouth with water. Get medical attention. Skin contact: In case of contact, immediately wash skin with soap and water for at least 15 minutes. Remove contaminated clothing. Wash clothing before reuse. Eye contact: Immediately flush eyes water for at least 15 minutes. Get medical attention.

V. Fire Fighting Measures: Flash Point: Data not available. Autoignition Temperature: Data not available. Explosion: Data not available. Fire extinguishing media: Water spray, dry chemical, foam, or carbon dioxide. Firefighting: Wear protective clothing and self-contained breathing apparatus to prevent contact with skin and eyes. May emit toxic fumes under fire conditions.

VI. Accidental Release Measures: Wear appropriate personal protective equipment. Sweep up material and avoid raising dust. Transfer to a closed chemical waste container for disposal. Wash spill site after material has been picked up for disposal.

VII. Handling and Storage: Store at 4°C in tightly closed container. Avoid inhalation of vapor or mist. Avoid contact with eyes, skin, and clothing. Wash thoroughly after handling.

© 2010 Cell Signaling Technology, Inc.

[email protected]

Labeling according to EC Directives: This product does not need to be labeled, in accordance with EC Directives or respective laws. US Regulatory Information: SARA Listed: No Canada (WHMIS): DSL No, NDSL No.

XVI. Other Information:

This compound is sold only for research use only. It is not for use in humans. To the best of our knowledge, this document is accurate. It is intended to serve as a guide for safe use of this product in a laboratory setting by experienced personnel. The burden of safe use of this material rests entirely with the user. Cell Signaling Technology, Inc., shall not be held liable for any damage resulting from the handling of or from contact with the above product.

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Material Safety Data Sheet (MSDS) for Cell Lysis Buffer (10X)

VIII. Exposure Controls/Personal

IX. Physical and Chemical Properties

Product name: Cell Lysis Buffer (10X) Product Catalog: 9803 CAS number: Not applicable to mixtures Manufacturer Supplier: Cell Signaling Technology 3 Trask Lane Danvers, MA 01923 USA 1-978-867-2300 TEL 1-978-867-2400 FAX 1-978-578-6737 Emergency Phone

IX. Physical and Chemical Properties

Appearance: Odor: pH: Boiling Point: Melting or Freezing Point: Flash Point: Volatile Organic Compounds (VOC): Autoignition temp. Solubility (water):

Clear liquid Data not available Data not available Data not available Data not available Data not available Data not available Data not available Soluble in water

This product is For Research Use Only. According to 29 CFR 1910.1200(d), mixtures with hazardous ingredients at less than 0.05%

Emergency Overview: Warning: May cause eye, skin, and respiratory tract irritation.

XII. Ecological Information: No data available.

US Regulatory Information: SARA Listed: No Canada (WHMIS): DSL No, NDSL No. EU Classification: Hazard Code: Xn = Harmful Risk Statement: 22: Harmful if swallowed 36/37/38 = Irritating to eyes, skin, respiratory tract Safety Statement: 26 = In case of contact with eyes, rinse immediately with plenty of water and seek medical advice. 28 = After contact with skin, wash immediately with plenty of water 37 = Wear suitable gloves

CAS# 7732-18-5 54827-17-7

FOR RESEARCH USE ONLY. NOT FOR USE IN HUMANS.

2400 mg/kg, mouse 708 mg/kg, rat 1560 mg/kg, rabbit >720mg/m3/1H, rat

XVI. Other Information:

VIII. Exposure Controls/Personal:

Orders n 877-616-CELL (2355)

Ingredient Name Water 3,3’,5,5’-tetramethylbenzidine (non-carcinogenic analog of benzidine) Proprietary formulation (non-hazardous)

No component of this product presensts at level greater than or equal to 0.1% is identified as a known or anticipated carcinogen by NTP, IARC, or OSHA.

Acute Skin Toxicity (LD50): Inhalation (LC50):

Reactivity: 0

IV. First Aid Measures:

IX. Physical And Chemical Properties:

II. Composition/Information on Ingredients:

XI. Toxicological Information: Proprietary Ingredient: Acute Oral Toxicity (LD50):

Rev. 05/19/10

Product name: TMB Substrate Solution Product Catalog: 7004 CAS number: None Manufacturer Supplier: Cell Signaling Technology 3 Trask Lane Danvers, MA 01923 USA 1-978-867-2300 TEL 1-978-867-2400 FAX 1-978-578-6737 Emergency TEL

Stability: Chemically stable under ordinary conditions of usage. Conditions to avoid: Heat. Materials to avoid: Strong oxidizing agents, reducing agents, bases. Hazardous Decomposition: Carbon monoxide, carbon dioxide. Hazardous polymerization: Will not occur.

FOR RESEARCH USE ONLY. NOT FOR USE IN HUMANS.

NFPA Hazard Classification: Health: 2 Flammability: 0

clear liquid none no data available no data available no data available no data available no data available Not flammable Not an oxidizer data not available >2.00 soluble in water

X. Stability and Reactivity:

III. Hazard Identification:

Material Safety Data Sheet (MSDS) for TMB Substrate Solution

I. Identification:

IX. Physical And Chemical Properties:

Product name: STOP Solution

© 2010 Cell Signaling Technology, Inc.

#7004 MSDS

Rev. 5/19/10

VI. Accidental Release Measures:

Wear appropriate personal protective equipment as indicated in Section VIII. Absorb liquid with an absorbent material. Transfer contaminated absorbent to a closed chemical waste container for disposal. Wash spill site after material has been picked up for disposal.

VII. Handling And Storage:

Store at 2°C – 8°C in tightly closed container. Avoid light and elevated temperatures. Avoid contact with eyes, skin, and clothing, wash thoroughly after handling. © 2010 Cell Signaling Technology, Inc.

#7002 MSDS

Material Safety Data Sheet (MSDS) for STOP Solution

VIII. Exposure Controls/Personal : Ventilation System: a system of local and/or general exhaust is recommended. Skin Protection: wear compatible chemical resistant gloves and protective clothing. Eye protection: wear protective safety glasses or chemical safety goggles. Maintain eye wash fountain and quick-drench facilities in work area.

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Appearance: Odor: Boiling Point: Melting Point: Freezing Point: Volatile Organic Compounds (VOC): Autoignition temp. Flammability Oxidizing Properties Specific Gravity: pH: Solubility:

clear to light yellow liquid none no data available no data available no data available no data available no data available not flammable not an oxidizer 1.010 (H20 = 1.0) 3.35-3.75 soluble in water

X. Stability and Reactivity: Stability: Chemically stable under ordinary conditions of usage. Conditions to avoid: Light, elevated temperatures and moisture. Materials to avoid: Strong oxidizing agents and metals. Hazardous Decomposition: May lead to carbon monoxide, carbon dioxide and nitrogen oxide. Hazardous polymerization: Will not occur.

XI. Toxicological Information: To the best of our knowledge, the chemical, physical, and toxicological properties of this buffer solution have not been thoroughly investigated. Routes of Exposure: Unknown at this time. Avoid ingestion, inhalation, contact with skin, eyes. Signs and Symptoms of Exposure: Unknown at this time.

XII. Ecological Information: No data available. XIII. Disposal Considerations: Dispose of in accordance with fed-

eral, state and local environmental regulations. Contact a licensed professional waste disposal service to dispose of this material.

XIV. Transport Information:

D.O.T. Proper Shipping Name: None This substance is considered non-hazardous for transport. IATA Proper Shipping Name: None This substance is considered non-hazardous for air transport.

XV. Regulatory Information:

US Regulatory Information: SARA Listed: No Canada (WHMIS): DSL No, NDSL No. EU Regulations Information: Not available for this chemical substance. EU Classification: (FOR 3,3’,5,5’-tetramethylbenzidine, CAS#54827-17-7) Hazard Code: Xi = Irritant Risk Statement: 36-38 = Irritating to eyes, respiratory system and skin.

XVI. Other Information:

This product is not intended for use in humans. To the best of our knowledge, this document is accurate. It is intended to serve as a guide for safe use of this product in a laboratory setting by experienced personnel. The burden of safe use of this material rests entirely with the user. The above information is believed to be accurate but is not necessarily all-inclusive and shall be used only as a guide. Cell Signaling Technology, Inc., shall not be held liable for any damage resulting from the handling of or from contact with the above product.

Support n 877-678-TECH (8324)

[email protected]

Web n www.cellsignal.com