Phenotype-specific immunodetection of cyclins using 488/630 nm dual laser flow cytometry

William Telford Hospital for Special Surgery

• • This protocol is for use with the D and E cyclins and employs 488 nm argon laser excitation of propidium iodide and a FITC-conjugated phenotypic label, and 630 nm NeNe or diode laser excitation of the fluorochrome Cy5 to detect cell cycle-specific cyclin D expression. Unlike previously used below 0°C fixation protocols, this method employs a gentle ethanol fixation step, preserving surface marker expression and immunolabeling. 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. It can also be used with other cyclin antibodies (such as those against cyclin A and B1) and antibodies against proliferating cell nuclear antigen (PCNA) shown to be detectable with a single ethanol fixation step. 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)
  • FITC-conjugated antibody against surface marker of interest 0.1% paraformaldehyde in PBS
  • 70% EtOH in glycerol (stored at 4°C)
  • staining buffer (1% BSA in PBS with 0.005% Tween 20 and 0.1% sodium azide)
  • propidium iodide (50 m 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 with 0.1% sodium azide.  Label with the FITC-conjugated antibody of interest in cold PBS with 2% FBS and 0.1% sodium azide.
• Wash cells once with PBS/FBS/azide and once with PBS/azide alone.  Decant the supernatant, shake tube gently to resuspend pellet and add 1.0 ml cold 0.1% paraformaldehyde in PBS.  Incubate for at least two hours or overnight at 4oC.
• Wash the cells twice with cold PBS/azide and place on ice.  Add 2 mls 70% EtOH in glycerol that has been kept at 4°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 µl.  Incubate overnight at 4°C.
• Wash twice with cold staining buffer and add the Cy5-conjugated anti-mouse IgG (available from Jackson ImmunoResearch and Caltag) in a volume of 200 µl.  Incubate for 4 hours at 4°C.
• Wash once with cold staining buffer and once  with cold PBS/azide.  Resuspend the cells in propidium iodide at 50 µg/ml in PBS with 100 U/ml RNase.  Analyze on any 488 nm argon- 630 nm HeNe or diode dual laser flow cytometer for FITC, PI and Cy5 fluorescence.  Both PI and Cy5 should be analyzed in linear mode.

• This technique has been used successfully to detect cyclin expression in several tumor cell lines, activated human lymphocytes and chick chondrocytes.

Below, HL-60 cells labeled for cyclin D1 either with 80% EtOH treatment at �20°C or 70% EtOH at 4°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 .