This is a cached page for the URL (http://www.scienceboard.net/resources/protocols.asp?action=article&protocol_id=49). To see the most recent version of this page, please click here.
Protocol Online is not affiliated with the authors of this page nor responsible for its content.
About Cache
The Science Advisory Board - Protocols, Product Reviews, Member Forum, and Science News
Note: You are seeing this message either because your browser has not loaded our stylesheets, or because your browser does not support stylesheets (CSS). Please upgrade to a relatively modern browser to improve your experience. Not sure what to upgrade to? Try Firefox.
The Science Advisory Board
Screen Name: 
 
Password: 
 

Protocols

Welcome to The Science Advisory Board's extensive database of research protocols for the biomedical sciences. Protocols are organized by techniques and are fully searchable by key word(s). Protocols are divided into a brief description of the methods, a step-by-step overview of the procedure, a collection of recipes, the supplies needed, and any helpful tips. Members submitting protocols will be rewarded through the ViewPoints system.
... back to articles list

Immunodetection of cyclin D1 and D2/D3 using flow cytometry

Posted on Saturday, October 18, 2003

Description
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

Procedure
Prepare the cell type of interest as a single cell suspension and wash once with cold PBS Decant the supernatant, shake tube gently to resuspend pellet and add 1.0 ml cold 0.5% paraformaldehyde in PBS. Incubate for at least two hours or overnight at 4C.
Wash the cells twice with cold PBS/azide and place on ice. Add 2 mls 80% EtOH in ddH20 that has been kept -20C. Incubate the cells for at least two hours. Wash once with staining buffer and decant.
Add the primary anti-cyclin antibody in a volume of 200 l. Incubate overnight at 4C.
Wash twice with cold cyclin staining buffer and add the Cy5-conjugated anti-mouse IgG (available from Jackson ImmunoResearch and Caltag) in a volume of 200 l. Incubate for 4 hours at 4C
Wash once with cold cyclin staining buffer and once with cold PBS/azide. Resuspend the cells in propidium iodide at 50 g/ml in PBS with 100 U/ml RNase. Analyze on any 488 nm argon- 630 nm HeNe or diode dual laser flow cytometer for PI and Cy5 fluorescence. Both PI and Cy5 should be analyzed in linear mode.


Recipes
Anti-cyclin D1 (Pharmingen cat. no. 14561A, clone G12-4326) or cyclin D2/D3 (cat. no. 14711A, clone G107-22). Other cyclin antibodies may be appropriate as well.
Cy5-conjugated anti-mouse IgG (Jackson ImmunoResearch and Caltag)
0.5% paraformaldehyde in PBS
80% EtOH in ddH20 (stored at -20C)
cyclin staining buffer (1% BSA in PBS with 0.005% Tween 20 and 0.1% sodium azide)
propidium iodide (50 g/ml solution with 100 U/ml DNase-free RNase)


Supplies


Tips


Submitted by: manny440

Scientific & Medical
Experts Needed!

The Science Advisory Board is the world's most established network of life scientists!

Voice your opinions on companies, products, protocols and even humor in a lively, real-time, interactive Online Community of over 42,000 life science & medical professionals.

Redeem generous rewards for participation in studies, contributing website content and referring colleagues.

Join Right Now!
(It's Free!)

Search This Site
only search scienceboard.net
only search Forums
What's this?