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).
