I have performed cell cycle analysis with this reagent following the protocol provided, however the output was not as expected. I used normal cells without any treatment so the typical G0/G1, S, and G2/M phases should be clearly present. Any idea what could have went wrong? Thanks in advance.
Hi..
From the look of your graph, it appears as if you are getting a highly mixed population. Also, you have quite some cells in the sub G0 phase. Neither is the S phase or the G2-M phase distinctly visible. Also it appears that you got a lot of background in your sample.
I would suggest, rather recommend, you following the protocol that I generally use. I would also suggest running this protocol first on any control cell line and not directly on stem cells so as to be sure that the protocol and the FxCycle is working just fine.
Anyway, I would want you to try out the following protocol as to what I use and works absolutely great.
Prepare 70% ethanol and store it in a freezer (0 deg or -20 deg) before starting the procedure.
1. Harvest cells (300,000 to 500,000 max; do not exceed this number as it generally tends to increase the CV and you'd get broad peaks) by trypsinization.
2. Centrifuge at 1200rpm for 3 minutes.
3. Give 1 PBS wash at 1200rpm for 3 minutes.
4. Fix your cells with 1ml of the 70% ethanol you made above, pipette, and store them at -20 deg C for overnight.
NOTE: Add 70% ethanol drop wise and slowly through the walls of the tube avoiding any clump formation. It can be stored in -20 deg C for atleast a month.
5. Next day, centrifuge at 1200rpm for 3 minutes.
6. Slowly decant the ethanol taking care the cells are not lost during this process as the cells become very loose due to the presence of ethanol.
7. Give 1-2 PBS wash(es) depending on the pellet and the cell loss.
8. Transfer the cells to the FACS tube and add the recommended amount of your FxCycle PI solution.
NOTE: Since this solution already contains RNAse A (as per what I read in the data sheet), prior incubation with RNAse A is not required. Since PI binds to RNA also along with the DNA, inhibition/inactivation of RNA becomes a necessary step.
9. Incubate at 37 deg C for 15-30 minutes.
10. Proceed with Acquisition.
Hope this will help.
Feel free to ask if you have any more questions.
Good luck!
Amir
Hello Nareshwaran,
Could you shed more light on your output so it becomes easier to tackle the exact problem that you faced?
Thanks.
Amir
Hello.. I am trying to understand the cell cycle profile of stem cells which were isolated from dental pulp. I followed the protocol and used 4% PFA to fix the cells prior to mixing with the said reagent. But the output which I obtained were as attached below.
I am not sure what else I left out. Thanks.
Hi..
From the look of your graph, it appears as if you are getting a highly mixed population. Also, you have quite some cells in the sub G0 phase. Neither is the S phase or the G2-M phase distinctly visible. Also it appears that you got a lot of background in your sample.
I would suggest, rather recommend, you following the protocol that I generally use. I would also suggest running this protocol first on any control cell line and not directly on stem cells so as to be sure that the protocol and the FxCycle is working just fine.
Anyway, I would want you to try out the following protocol as to what I use and works absolutely great.
Prepare 70% ethanol and store it in a freezer (0 deg or -20 deg) before starting the procedure.
1. Harvest cells (300,000 to 500,000 max; do not exceed this number as it generally tends to increase the CV and you'd get broad peaks) by trypsinization.
2. Centrifuge at 1200rpm for 3 minutes.
3. Give 1 PBS wash at 1200rpm for 3 minutes.
4. Fix your cells with 1ml of the 70% ethanol you made above, pipette, and store them at -20 deg C for overnight.
NOTE: Add 70% ethanol drop wise and slowly through the walls of the tube avoiding any clump formation. It can be stored in -20 deg C for atleast a month.
5. Next day, centrifuge at 1200rpm for 3 minutes.
6. Slowly decant the ethanol taking care the cells are not lost during this process as the cells become very loose due to the presence of ethanol.
7. Give 1-2 PBS wash(es) depending on the pellet and the cell loss.
8. Transfer the cells to the FACS tube and add the recommended amount of your FxCycle PI solution.
NOTE: Since this solution already contains RNAse A (as per what I read in the data sheet), prior incubation with RNAse A is not required. Since PI binds to RNA also along with the DNA, inhibition/inactivation of RNA becomes a necessary step.
9. Incubate at 37 deg C for 15-30 minutes.
10. Proceed with Acquisition.
Hope this will help.
Feel free to ask if you have any more questions.
Good luck!
Amir
Thank you very much, Mr. Amir.. I will definitely try your suggested protocol and will update you on the outcome. Thanks once again. =)
Dear Mr. Amir,
I have tried the method as you have suggested. Now the results were better and I could get a comparable pattern as suggested by manufacturer.
Thanks you so much. =)
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