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Identification of cells in S phase using the Cell Proliferation Fluorescence Kit and IN Cell Analyzer 1000


The cell cycle is of key importance to many areas of drug discovery. On the one hand this fundamental process provides the opportunity to discover new therapeutic targets for anti-cancer agents, and on the other hand drugs and targets in other therapeutic areas must be tested for undesirable effects on the cell cycle.

The Cell Proliferation Fluorescence Kit is a precise, fast, and simple fluorescence assay to quantitate cell proliferation, study the regulation of DNA synthesis, or observe the degree of inhibition of cell progression through S phase. It is based on the measurement of 5-bromo-2'-deoxyuridine (BrdU) incorporation during DNA synthesis in proliferating cells.

BrdU (an analog of the DNA precursor thymidine) is incorporated into newly synthesized DNA by cells entering and progressing through the S (DNA synthesis) phase of the cell cycle. The incorporated BrdU is detected with a specific anti-BrdU monoclonal antibody, followed by a Cy™5 labeled antibody to mouse immunoglobulin, giving a fluorescent signal at sites of BrdU incorporation. This provides an excellent marker for identifying cells that are in S phase, and also for determining the proportion of cells in S phase of the cell cycle.

The intensity of nuclear-associated Cy5 fluorescence is measured using an IN Cell Analyzer 1000 and the data analyzed using an Object Intensity Analysis Module.

The use of a Cy5 red-shifted fluor enables this technology to be multiplexed with green fluorescent protein (GFP). This assay provides a high-throughput method for the identification and analysis of cells that have actively synthesized DNA.


Materials
Products used

IN Cell Analyzer 1000 25-8010-26

Object Intensity Analysis Module 25-8010-56
for IN Cell Analyzer 1
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