< S. Coats 1995
Cytometry of Fibroblast Nuclei for DNA content
4 mM Na3Citrate (0.118 g/100 mL)
30 U/mL RNAseI (43
0.1% Triton-X100 (0.1mL/100 mL)
50 µg/mL propidium
iodide (5 mg/100 mL)
- 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.