I know cell cycle, how it works and we are looking for which step of cell cycle is affected after treating with a compound. This must be the reason for studying cell cycle analysis.
What I don't really get is how to relate this cell cycle with the amount of DNA that reveal in cell cycle analysis? I run two to three times and noticed that DNA in G0/G1 stage is highest, S phase is 2nd and M phase is lowest. This happened to non-treated cells as well as treated cell too. So what is this result? and what to expect in deed by doing this analysis? My cells are cancer cells. Is this normal to find DNA highest in G0/G1 for all type of cells?
Hello Jamail,
Did you plot PI (DNA content) on X-axis and Count (Intensity) on Y-axis? The count (intensity, Y-axis) will always be higher in G0/G1 phase as most of the cells remain in this phase than that of S and G2/M phase. But, DNA content (x-axis) must be double in G2/M phase than G1 phase as said by Antonios. Hope this helps!
Hi Subrata Pore and Antonios, I just understand basic of cell cycles in which what happens in steps. However, it comes to analysis and to interpret after flow result, what can I say. Also not sure it is correct and valid result or not? You can see my result in attach file. There you will see non treat and treated cells. DNA contents in G0/G1 always generally high compare to S and G2/M in both non treat and treat.
However, in comparison, treated one become more DNA in all phases. Notice that treated one also increased debris a lot too. If I am not wrong this is because of dead cells after treating with compound A which causes dead cells to debris later.
Can anyone help me see the attached results and how can we conclude from it and what can we say for that? Thanks.
There are some missing points for me to answer your question.
"Is this normal to find DNA highest in G0/G1 for all type of cells?"
Technically speaking, what you detect is fluorescence of the dye anchored to DNA. Having said so, the answer is yes. The major part of a cell population is in G0/G1 phase. This can change if you synchronize the cell culture (see link "18228388" for examples).
"This happened to non-treated cells as well as treated cell too. So what is this result? and what to expect in deed by doing this analysis?"
Compounds interact with biological systems in a dose-dependent and a time-dependent manner. Your results show that either
1) the dose or exposure time were too low, or
2) the compound does not interfere with the cell cycle.
(See attched pdf for possible effects)
My advice:
- Check which dose you are using. As a rule of thumb in my group for "unstudied" compounds, we expose cells to two drug doses for 24 h. These doses represent the GI50 and three-times the GI50 of said compound after exposure for 48 h.
- I highly recommend to use a standard drug as a control to check if you observe the expected changes in cell cycle
Hope this helps
http://www.ncbi.nlm.nih.gov/pubmed/18228388
Hello Jamail,
Your interpretation is wrong. I am not going to compare between treated and untreated results. First consider only untreated cells:
1) As I said earlier X-axis (DNA content index) gives you DNA content and Y-axis (count) gives you intensity. You are saying "DNA content in G0/G1 is always higher than other phases" which is NOT correct. Only intensity is higher of G0/G1 phase as number of cells are higher (count is higher) in that phase.
2) you wrongly marked different phases. Only BLUE part of graph (untreated cells) gives the whole picture of cell cycle. Very first peak is G0/G1 and 2nd peak is G2/M; whereas the valley between two peaks is S phase.
3) Now, you see the 1st peak maximum is around 1.4 (DNA content index) on x-axis and 2nd peak maximum is around 2.8 on X-axis. This means DNA content of G2/M phase is double compared to G0/G1 and in between there is S phase population.
4) What you marked with brown and green colors and considered S and G2/M phases, could be debris OR multi-nucleated cells. Depending on cells, some cells have more nuclei and more DNA and they should appear at far right on X-axis (DNA content index).
Everything I have written here based on your untreated data. It is very difficult to explain in writings here. Hope this helps you.
Hi Subrata pore, I want to check again with you the guide i found is contradicted to what you were saying about brown and green color were junks. In deed they are marker too for phases of cell cycles.
You can read here link below as I was not able to attach the file. Page 7, point 6. saying:
Adjust all four markers to distinguish the G0/G1 (blue), S (purple), and G2/M (green) cells. Touch and drag the
individual markers, or touch a marker and use the arrow buttons to adjust the position. Be sure to exclude all
debris.
Link: https://www.google.com/webhp?sourceid=chrome-instant&ion=1&espv=2&ie=UTF-8#q=muse%20cell%20cycle%20user%20guide%20line
(first PDF file "Cell Cycle Kit")
I am confused because contradicted to each other.
Another example in one of the published paper. You can see cell cycles plots and this is different from what Subrata pore and Antonios mentioned. Confused about it. Are results different by FACS and MUSE?
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