Invitrogen™ Click-iT™ EdU Proliferation Assay for Microplates

Catalog No.	Quantity
C10499	400 Assays

Catalog No. C10499 Supplier Invitrogen™ Supplier No. C10499

Description

The Click-iT EdU Proliferation Assay for Microplates provides a more simplified and robust method for the measurement of mammalian cell proliferation compared to traditional BrdU-based methods.

After incorporation of EdU (a modified thymidine analogue) into newly synthesize DNA, horse radish peroxidase (HRP) is covalently attached via the highly specific click reaction. Amplex UltraRed Reagent, an HRP substrate, is then added and the resulting fluorescence signal detected. Because of the highly specific attachment of HRP to EdU, this assay results in highly sensitive and reliable measurement of mammalian cell proliferation in a microplate format.

Click-iT EdU Proliferation Assay for Microplates features include:
• Improved performance—direct conjugation of HRP to incorporated EdU improves assay sensitivity and accuracy
• Increased reliability—assay format results in low well-to-well variability
• Simplified protocol—results in reduced time to data; easier to follow protocol
• Multiplex enabled—assay can be easily multiplexed with other cell health reagents resulting in more data per sample

Measuring cell proliferation is a fundamental method of assessing cell health, determining genotoxicity, and evaluating anti-cancer drugs. The most accurate method of measuring proliferation is by direct detection of DNA synthesis. Originally this was accomplished through incorporation of a radioactive nucleoside (e.g., ³H-thymidine). This method was replaced by antibody-based detection of the nucleoside analog bromo-deoxyuridine (BrdU). After incorporation of BrdU into the newly synthesized DNA, denaturation is required to expose the BrdU molecules to the anti-BrdU antibody. The denaturation involves harsh methods (HCl, heat, or enzymes) and is time consuming and difficult to perform consistently.

The Click-iT EdU Proliferation Assay is an alternative to the BrdU assay. The nucleoside analog EdU (5-ethynyl-2´-deoxyuridine) is added to live cells and incorporated into DNA during active DNA synthesis. The incorporated EdU contains an alkyne group which is covalently joined to the azide group present in HRP using a highly specific copper-catalyzed covalent reaction ('click reaction'). Amplex UltraRed Reagent is then added and its conversion by HRP into a highly fluorescent product is recorded using a fluorescence microplate reader (excitation: 568, emission: 585 nm). With a high extinction coefficient, good quantum efficiency, and resistance to auto-oxidation, Amplex UltraRed Reagent delivers higher sensitivity and a broader assay range than other fluorogenic or colorimetric HRP substrates. It also offers versatility through detection by either fluorescence or absorbance measurement.

Specifications

• Content And Storage: Store at -20°C, desiccated and protected from light.
• Detection Method: Fluorescence
• For Use With (Application): Proliferation Assay
• For Use With (Equipment): Microplate Reader
• Product Type: Proliferation Assay
• Format: 96-well plate
• Green Features: Less hazardous
• Product Line: CyQUANT
• Quantity: 400 Assays
• Shipping Condition: Approved for shipment at Room Temperature or on Wet Ice

Frequently Asked Questions (FAQs)

I am not getting any signal with the Click-iT EdU Cell Proliferation Assay for Microplates. What can cause this?

If the Amplex UltraRed reagent was spontaneously oxidized, all samples would have high levels of signal. The lack of any signal indicates that some component in the reaction is non-functional or little or no EdU was incorporated by the cells. The HRP-azide (Component B), the Click-iT EdU reaction additive (Component F), and the hydrogen peroxide (Component J) are the most labile components in the kit. HRP can be denatured and inhibited by sodium azide. The Click-iT Edu reaction additive (Component F) should be diluted only shortly before use and dilute solutions should not be stored. Hydrogen peroxide does spontaneously degrade over time, but more readily when diluted; prepare the hydrogen peroxide stock solution shortly before use and do not store.

With the Click-iT EdU Cell Proliferation Assay for Microplates, can we use another HRP substrate other than the Amplex UltraRed reagent provided in the kit?

This may be possible, but the final working concentrations and incubation steps would have to be optimized by the user. Be advised that chromogenic substrates are not as sensitive as the fluorogenic Amplex UltraRed reagent.

Can endogenous peroxidase activity interfere with the Click-iT EdU Cell Proliferation Assay for Microplates?

The Click-iT EdU Cell Proliferation Assay for Microplates protocol requires samples to be fixed and permeabilized prior to the click reaction. Most endogenous peroxidases may be inactivated after fixation/permeabilization and after the click reaction. If this is of concern, apply the Amplex UltraRed reaction mixture without added H2O2 to the samples after fixation, but before the click reaction (i.e., no HRP-azide added to the sample). In the absence of added H2O2 and HRP-azide, if you see any change in Amplex UltraRed fluorescence intensity, this is indicative of endogenous peroxidase activity.

For Research Use Only. Not for use in diagnostic procedures.