| 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.
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 .
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