Experimental Protocol:

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

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)

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.

This technique has been used successfully to detect cyclin D₁ expression in several tumor cell lines, activated human lymphocytes and chick chondrocytes. Below, HL-60 cells labeled for cyclin D1 or cyclin D2/D3 with 80% EtOH treatment at –20°C.

References

Gong, J., Bhatia, U., Traganos, F. and Darzynkiewicz, Z. 1995. Expression of cyclins A, D2 and D3 in individual normal mitogen stimulated lymphocytes and in MOLT-4 leukemic cells analyzed by multiparameter flow cytometry. Leukemia 9, 893-899.

Juan G., Gong, J., Traganos, F. and Darzynkiewicz, Z. 1996. Unscheduled expression of cyclins D1 and D3 in human tumour cell lines. Cell Prolif. 29, 259-266 .

CD-ROM Vol 4 was produced by Monica M. Shively and other staff at the Purdue University Cytometry Laboratories and distributed free of charge as an educational service to the cytometry community. If you have any comments please direct them to Dr. J. Paul Robinson, Professor & Director, PUCL, Purdue University,West Lafayette, IN 47907, Phone: (765)-494-0757 FAX(765) 494-0517, Web http://www.cyto.purdue.edu , EMAIL cdrominfo@flowcyt.cyto.purdue.edu