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DNA Analysis Protocols
 

This material was originally published in the Purdue Cytometry CD-ROM Series,volume 4


Immunodetection of cyclin D1 and D2/D3 using 488/630 nm dual laser flow cytometry

 
William Telford
Hospital for Special Surgery



This protocol is for use with the D cyclins and employs 488 nm argon laser excitation of propidium iodide and 630 nm NeNe or diode laser excitation of the fluorochrome Cy5 to detect cell cycle-specific cyclin D expression. It has been tested with antibodies against cyclin D1 (Pharmingen cat. no. 14561A, clone G12-4326), cyclin D2/D3 (cat. no. 14711A, clone G107-22) and cyclin E, and is based on protocols originally designed by Z. Darzynkiewicz and colleagues. Although FITC-conjugated secondary or direct FITC-conjugated anti-cyclin antibodies can also be used, the low autofluorescence background seen with 630 nm laser excitation makes Cy5 detection of cyclin expression particularly sensitive. Using dual laser excitation also eliminates the need for fluorescence compensation. This assay can be used with any instrument employing dual laser excitation, including the B-D Vantage, FACSCalibur and Coulter Elite.

Materials

 

 

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