Phenotype-specific immunodetection of cyclins using488/630 nm dual laser flow cytometry
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.
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
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%
propidium iodide (50 m g/ml solution
with 100 U/ml DNase-free RNase)
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
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.
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. Leukemia9, 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 .