Protocol:
Preparation of Samples for Cell Cycle Analysis by Flow Cytometry:
1) Suspend approximately million cells (10^6) in 1mL of phosphate-buffered saline solution (1x). And with the help of pipette, gently aspirate about 7-8 times, to make the single cell suspension.
2) Take a centrifuge tube and add 4mL of 70% ethanol. (70% ethanol should be one ice before using). and add 1mL of cells in PBS to this tube. Keep the tubes on ice for minimum 2 hours at 4℃.. This step will fix the cells. (cells can kept at 4℃ for a week).
3) Centrifuge the ethanol fixed cells for 5 minutes at 300g. Carefully take out the supernatant. Do not disturb the pellet.
4) Add 5mL of cold 1x PBS to this pellet and suspend the cells using pipette.
5) Centrifuge the tubes at 300g for 5 minutes. and discard the supernatant. Again do not disturb the pellet.
6) if required, step 5 can be repeated for one more time.
7) Add 1mL of Propidium Iodide (PI) Solution to the pellet and suspend the pellet. (PI concentration should be 50µg/mL).. (Additionally, you can add 100µg/mL RNase A before adding PI).
8) Keep the tubes at room temperature for at least 30 minutes in the dark. (You can keep the tubes at 37℃ for 8-10 minutes to speed up things).
9) Transfer PI stained cells to flow cytometer tubes and run the flow cytometer for the cell cycle analysis.
10) For the excitation of PI, 488nm or 561nm lasers can be used and emission at 620nm.
Emission can be measured using Long Pass 600nm filter or 610/20 band pass filter.
Tips:
1) Be sure to use approximately equal number of cells in each tubes. Better to count the cells before proceeding.
2) Try to get a good single cell suspension for better staining and better results.
3) Use proper and equal PI concentration for each samples.
Example of how cell cycle profile looks in a flow cytometry data.:
Preparation of Samples for Cell Cycle Analysis by Flow Cytometry:
1) Suspend approximately million cells (10^6) in 1mL of phosphate-buffered saline solution (1x). And with the help of pipette, gently aspirate about 7-8 times, to make the single cell suspension.
2) Take a centrifuge tube and add 4mL of 70% ethanol. (70% ethanol should be one ice before using). and add 1mL of cells in PBS to this tube. Keep the tubes on ice for minimum 2 hours at 4℃.. This step will fix the cells. (cells can kept at 4℃ for a week).
3) Centrifuge the ethanol fixed cells for 5 minutes at 300g. Carefully take out the supernatant. Do not disturb the pellet.
4) Add 5mL of cold 1x PBS to this pellet and suspend the cells using pipette.
5) Centrifuge the tubes at 300g for 5 minutes. and discard the supernatant. Again do not disturb the pellet.
6) if required, step 5 can be repeated for one more time.
7) Add 1mL of Propidium Iodide (PI) Solution to the pellet and suspend the pellet. (PI concentration should be 50µg/mL).. (Additionally, you can add 100µg/mL RNase A before adding PI).
8) Keep the tubes at room temperature for at least 30 minutes in the dark. (You can keep the tubes at 37℃ for 8-10 minutes to speed up things).
9) Transfer PI stained cells to flow cytometer tubes and run the flow cytometer for the cell cycle analysis.
10) For the excitation of PI, 488nm or 561nm lasers can be used and emission at 620nm.
Emission can be measured using Long Pass 600nm filter or 610/20 band pass filter.
Tips:
1) Be sure to use approximately equal number of cells in each tubes. Better to count the cells before proceeding.
2) Try to get a good single cell suspension for better staining and better results.
3) Use proper and equal PI concentration for each samples.
Example of how cell cycle profile looks in a flow cytometry data.:
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