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Reagents

Download the EZ-BrdU Protocol Booklet here.

The Phoenix Flow Systems, Inc. EZ-BrdU (cat # AC-1001) assay kit is a two color staining method for measuring cell proliferation by multi-parameter analysis of DNA replication and cellular DNA content/cell cycle position with flow or image cytometry (2). The kit contains instructions and enough reagents for 50 assays which includes the BrdU solution for feeding to the cells before they are stained, washing, rinsing buffers for processing individual steps in the assay; denaturation, neutralization buffers for making the incorporated BrdU accessible for labeling, fluorescein labeled anti-BrdU antibody (F~PRB-1) for labeling incorporated DNA, and propidium iodide/RNase A solution for counter staining the total DNA. In addition, positive control cells are included to eliminate the staining variable of the assay. The positive control cells are ethanol fixed cultured cells which have BrdU incorporated into their DNA. Phoenix Flow Systems also offers other dye configurations for the assay including TRITC, and the Alexa dyes from Invitrogen.

This cell proliferation kit is optimized for measuring intracellular markers simultaneously with cell proliferation. When used in this configuration, an Alexa 647 conjugation of our anti-BrdU should be requested when ordering the kit. These experiments will also require access to a two laser flow cytometer because the Alexa 647 is excited with a HeNe laser. By using this configuration, the green fluorescent detector channel of the flow cytometer is available for measuring cyclins or mpm-2 (mitotic marker) For a publication employing this method, click here.
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cell proliferation

FLOW DIAGRAM OF

EZ--BrdU ASSAY

Contents of the EZ-BrdU Kit:

Positive Control Cells

BrdUrd Cell Feeding Solution

Wash Buffer

Rinse Buffer

Denaturation Buffer

Neutralization Buffer

Fluorescein-anti BrdU antibody

Propidium Iodide/RNase DNA staining solution