ImmunodetectionofcyclinD1andD2／D3usingflowcytometry

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 4°C. 
Wash the cells twice with cold PBS/azide and place on ice. Add 2 mls 80% EtOH in ddH20 that has been kept -20°C. 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 μ l. Incubate overnight at 4°C. 
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 μ l. Incubate for 4 hours at 4°C 
Wash once with cold cyclin staining buffer and once with cold PBS/azide. Resuspend the cells in propidium iodide at 50 μ 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 -20°C) 
cyclin staining buffer (1% BSA in PBS with 0.005% Tween 20 and 0.1% sodium azide) 
propidium iodide (50 μ g/ml solution with 100 U/ml DNase-free RNase)