DNA STAINING OF ISOLATED NUCLEI FOR CELL CYCLE ANALYSIS

I. SAMPLE - one or more of the following preparations

A.  Suspension of single cells from tissue (e.g., lymph node, spleen, bone marrow, placental cells)

B.  Tissue culture cells

C.  Ficoll-hypaque separated mononuclear cells

II. REAGENT - hypotonic staining buffer for DNA

We have kept this solution in a tightly-sealed bottle protected from light for several months without apparent loss of staining activity.

III. STAINING

1. Place 1x10⁶ cells into each tube.

2. Spin down samples and remove supernatant as completely as possible without disturbing the pellet.

3. Add 1 ml of the hypotonic DNA staining buffer to the pellet and mix well.

4. Keep samples at 4^°C protected from light for 30 min or for a maximum of 1 h before acquisition on the flow cytometer. Note: Prolonged exposure to the hypotonic buffer can lead to an increase of debris in the sample.

Reference: Krishan A: Rapid flow cytofluorometric analysis of mammalian cell cycle by propidium iodide staining. J. Cell Biol. 66:188-193, 1975.

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