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
2 X 75 mm culture tubes
Vortex mixer
Waterbath at 37oC
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 2-8oC.
3. Remove supernatant by aspiration or rapid decanting and add 500 ml of cold PBS to the cell pellet. Mix gently. Add 500 ml of cold, buffered 2% formaldehyde solution and mix again. Incubate at 2-8oC for 1h.
Permeabilize cells with ethanol
4. Spin cells down by centrifugation for 5 min at 300 x g at 2-8oC, remove supernatant by aspiration or rapid decanting, wash once with cold 1 X PBS, then add 1 ml of 70% ethanol at – 20 oC drop-wise to the cell pellet with the tube sitting on a vortex. Incubate cell suspension overnight at 2-8oC.
5. Spin cells down by centrifugation for 5 min at 300 x g at 2-8oC, 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 37oC 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 70oC 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 2-8oC protected from light. The solution is stable for at least 1 month. Check pH periodically. Do not heat the solution above 70oC. For best results, use only very pure preparations of paraformaldehyde (i.e., electron microscopy grade from Polysciences).
References
Chu, YW, Wang R., Schmid I, Sakamoto KM. Analysis with flow cytometry of green fluorescent protein expression in leukemic cells. Cytometry 36:333-339, 1999.
Schmid I. and Sakamoto KM. Analysis of DNA content and green fluorescent protein expression. In: Current Protocols in Cytometry, Vol 1, Robinson JP, Darzynkiewicz Z, Dean P, Orfao A, Rabinovitch P, Stewart C, Tanke H, Wheeless L, eds., John Wiley & Sons, 2001, pp. 7.16.1-7.16.10.
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
2 X 75 mm culture tubes
Vortex mixer
Waterbath at 37oC
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 2-8oC.
3. Remove supernatant by aspiration or rapid decanting and add 500 ml of cold PBS to the cell pellet. Mix gently. Add 500 ml of cold, buffered 2% formaldehyde solution and mix again. Incubate at 2-8oC for 1h.
Permeabilize cells with ethanol
4. Spin cells down by centrifugation for 5 min at 300 x g at 2-8oC, remove supernatant by aspiration or rapid decanting, wash once with cold 1 X PBS, then add 1 ml of 70% ethanol at – 20 oC drop-wise to the cell pellet with the tube sitting on a vortex. Incubate cell suspension overnight at 2-8oC.
5. Spin cells down by centrifugation for 5 min at 300 x g at 2-8oC, 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 37oC 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 70oC 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 2-8oC protected from light. The solution is stable for at least 1 month. Check pH periodically. Do not heat the solution above 70oC. For best results, use only very pure preparations of paraformaldehyde (i.e., electron microscopy grade from Polysciences).
References
Chu, YW, Wang R., Schmid I, Sakamoto KM. Analysis with flow cytometry of green fluorescent protein expression in leukemic cells. Cytometry 36:333-339, 1999.
Schmid I. and Sakamoto KM. Analysis of DNA content and green fluorescent protein expression. In: Current Protocols in Cytometry, Vol 1, Robinson JP, Darzynkiewicz Z, Dean P, Orfao A, Rabinovitch P, Stewart C, Tanke H, Wheeless L, eds., John Wiley & Sons, 2001, pp. 7.16.1-7.16.10.
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