Flow Cytometry of Fibroblast Nuclei for DNA content

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)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.