Measurement of GFP Expression and DNA Content in Permeabilized Cells

Reagents

Cells to be studied expressing green fluorescent protein (GFP). Note that the same cell type without GFP is needed as a control.

1 X PBS

2% Buffered formaldehyde solution (see recipe)

70% Ethanol

Propidium iodide stock solution (1mg/ml in PBS)

DNAse-free ribonuclease A

12 X 75 mm culture tubes

Vortex mixer

Waterbath at 37o C

Method

Fix cells with formaldehyde

1. Count cells.

2. Place approximately 106 cells into a 12 x 15 mm test tube and wash them once with PBS by centrifugation for 5 min at 300 x g at 2-8o C.

3. Remove supernatant by aspiration or rapid decanting and add 500 m l of cold PBS to the cell pellet. Mix gently. Add 500 m l of cold, buffered 2% formaldehyde solution and mix again. Incubate at 2-8o C for 1h.

Permeabilize cells with ethanol

4. Spin cells down by centrifugation for 5 min at 300 x g at 2-8o C, remove supernatant by aspiration or rapid decanting, wash once with cold 1 X PBS, then add 1 ml of 70% ethanol at � 20 o C drop-wise to the cell pellet with the tube sitting on a vortex. Incubate cell suspension overnight at 2-8o C.

5. Spin cells down by centrifugation for 5 min at 300 x g at 2-8o C, remove supernatant by aspiration or rapid decanting and add 1 ml of a solution containing 40µg/ml of PI and 100 µg/ml of ribonuclease A. Incubate cell suspension at 37o C in the dark for 30 min. If needed, filter samples through a nylon mesh to remove clumps before acquisition on the flow cytometer.

Preparation of Buffered 2% Formaldehyde Solution

Add 2g paraformaldehyde (Polysciences, Inc.) to 100 ml PBS. Heat the solution to 70o C in a fume hood until the paraformaldehyde goes into solution (approximately 1 h ). Cool to room temperature, check pH and adjust to 7.2 with 0.1M NaOH or 0.1M HCl. Store at 2-8o C protected from light. The solution is stable for at least 1 month. Check pH periodically. Do not heat the solution above 70o C. For best results, use only very pure preparations of paraformaldehyde (i.e., electron microscopy grade from Polysciences).