Thermo Fisher Scientific Click-iT Plus EdU Alexa Fluor 350 Flow Cytometry Assay Kit

Invitrogen™ Click-iT™ Plus EdU Alexa Fluor™ 350 Flow Cytometry Assay Kit

The Click-iT Plus EdU Alexa Fluor 350 Flow Cytometry Assay Kit provides a simplified, robust assay for analyzing DNA replication in proliferating cells compared to traditional BrdU methods. Newly synthesized DNA is analyzed using the UV laser of the flow cytometer. The Click-iT Plus formulation is compatible with standard fluorophores, including R-PE and R-PE tandems, as well as fluorescent proteins.

주요 특징

• Multiplexable — compatible with R-PE (and tandems) and fluorescent proteins
• Accurate — superior results compared to BrdU assays
• Fast — results in as little as 60 minutes

Multiplexable

Click-iT Plus Alexa Fluor 350 EdU assays can be used in conjunction with R-PE and R-PE tandems, as well as fluorescent proteins such as GFP and mCherry.
The Alexa Fluor 350 labeling reagent is excited readily at 350 nm with a UV laser and emits at 438 nm.

Advanced Method Superior to BrdU

The Click-iT Plus EdU Flow Cytometry assay is a novel alternative to the BrdU assay.
EdU (5-ethynyl-2´-deoxyuridine) is a thymidine analog incorporated into DNA during active DNA synthesis.
Detection is based on click chemistry—a copper-catalyzed covalent reaction between an azide and an alkyne.
In this application, the alkyne is found in the ethynyl moiety of EdU, while the azide is coupled to the Alexa Fluor dye.
Standard flow cytometry methods are used to determine the percentage of S-phase cells in the population.

Mild Conditions Compatible with Cell Cycle Dyes and Antibodies

The small size of the dye azide enables efficient detection of incorporated EdU under mild conditions.
Standard aldehyde-based fixation and detergent permeabilization allow the Click-iT Plus detection reagent to access DNA.
Unlike BrdU assays that require harsh DNA denaturation (HCl, heat, or DNase), this assay preserves cell cycle distribution and antigen recognition sites.
Compatible with cell cycle dyes and multiplexing with antibodies against surface and intracellular markers, as well as conjugates labeled with R-PE, R-PE tandems, GFP, and mCherry.

Quick and Simple Protocol

The Click-iT Plus EdU protocol is based on aldehyde fixation and detergent permeabilization steps for immunohistochemical antibody labeling.
EdU is also compatible with saponin and methanol fixation/permeabilization agents.

5-Step Procedure

1. Treat cells with EdU
2. Fix and permeabilize cells
3. Detect S-phase cells with Click-iT Plus detection cocktail for 30 min
4. Wash once
5. Analyze

Results can be obtained in as little as 60 minutes, though 90 minutes is recommended for all applications.

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