Flow Cytometry of Fibroblast Nuclei for DNA content
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1104
Materials
P.I. Solution :
4 mM Na3Citrate (0.118 g/100 mL)
30 U/mL RNAseI (43 mg/100 mL)
0.1% Triton-X100 (0.1mL/100 mL)
50 µg/mL propidium iodide (5 mg/100 mL)
30 U/mL RNAseI (43 mg/100 mL)
0.1% Triton-X100 (0.1mL/100 mL)
50 µg/mL propidium iodide (5 mg/100 mL)
Procedure
- Harvest cells: Rinse with a subconfluent 10 mL dish with PBS (Ca++ /Mg++ free). Cover with 1.5 mL 0.1% (2.5 mM) EDTA in PBS at 37ºC x 10 min. Loosen cells by vigorous pipetting then transfer suspension to 1.5 mL Eppendorf tubes on ice.
- Spin at 1000 RPM, 4ºC. Discard supernatant. Resuspend in 0.5 mL PBS. Remove 0.1 mL for flow cytometry and use the remainder for protein extracts.
- Add 4 - 5 volumes of 100% EtOH and vortex gently. Incubate 15'. (The cells may be now stored at 4ºC.)
- Spin at 1000 RPM, and wash pellet with PBS.
- Resuspend pellet in 0.5 mL of P.I. solution (above). This should give ~1 million cells/mL. Incubate 10 min. at 37ºC.
- Filter through nylon mesh screen to remove cell clumps. Keep at 4ºC on the dark until ready for flow. See the Flow cytometer protocol for more details.
