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).
- Functional Analysis by Inducible RNA Interference in Drosophila melanogaster
- Gel Electrophoresis Assays for Analyzing DNA Double-Strand Breaks in Saccharomyces cerevisiae at Various Spatial Resolutions
- Generation of Protease-Deficient Strains and Their Use in Heterologous Protein Expression
- Preparation of Human Partial Chromosome Paints from Somatic Cell Hybrids
- Vertical Arrays: Microarrays of Complex Mixtures of Nucleic Acids
- Triplet Repeats and DNA Repair: Germ Cell and Somatic Cell Instability in Transgenic Mice
- 转基因――基因标靶
- 效应物(effector)
- MAPK ERK Pathway
- 细胞培养――细胞保藏