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 .