Hello Abhishek, I am not familiar with plant extracts but with FACS procedure. Therefore, the first thing which came into my mind: Did you count your cells and calculated your amount of PI per cell number in both samples (untreated and treated) the same way? The FL2 channel by a staining with PI will show you the DNA content since PI will intercalate with your DNA. In G1/G0 phase you will have less DNA per cell in contrast to a cell in S phase or even in G2/M phase before cell division since the DNA got doubled during S phase. Therefore, more PI molecules can intercalate within one cell with the DNA. In case you do not calculate your amount of added PI per cell number and stay the same between samples you would like to compare you will have the following: in each case your PI will not be adding in excess as usually the case for antibodies. Therefore, the amount of PI per cell number is limited. When you have more PI per cell number the peak will shift to the right because more PI molecules can intercalate with the DNA in ONE cell. I hope that helps and please let me know if you did that already in your former experiments. Best, Nicole

Its hard to say without seeing the raw data but my first guess is that you may have a spindle apparatus blocker activity and are inducing aneuploidy in your sample.

Hello Abhishek, I am not familiar with plant extracts but with FACS procedure. Therefore, the first thing which came into my mind: Did you count your cells and calculated your amount of PI per cell number in both samples (untreated and treated) the same way? The FL2 channel by a staining with PI will show you the DNA content since PI will intercalate with your DNA. In G1/G0 phase you will have less DNA per cell in contrast to a cell in S phase or even in G2/M phase before cell division since the DNA got doubled during S phase. Therefore, more PI molecules can intercalate within one cell with the DNA. In case you do not calculate your amount of added PI per cell number and stay the same between samples you would like to compare you will have the following: in each case your PI will not be adding in excess as usually the case for antibodies. Therefore, the amount of PI per cell number is limited. When you have more PI per cell number the peak will shift to the right because more PI molecules can intercalate with the DNA in ONE cell. I hope that helps and please let me know if you did that already in your former experiments. Best, Nicole

I agree with Nicole Forster, but would add that it is not the amount of PI per cell, but per cell and per DNA content. As PI intercalates into the DNA (or DNA of chromatin), it causes an unwinding of negative supercoiling. (The unwinding is a given number of degrees per intercallating molecule. This happens with all DNA intercallating agents). This has the effect of driving off histones (first H2A/H2b, and, at higher PI concentrations, then H3/H4, untill one has fully intercallated relaxed DNA. A strand break will increase the rate at which this happens.) and making more DNA available for PI intercallation. One needs to titrate the PI/DNA ratio for highly repeatable results.

@ Barry Barclay: Sir you are very right infact I was unable to properly describe the actual story, but there seems no way to upload any pictorial comments in researchgate....which I am missing a lot.

@ Nicole Foster: Thats a very good explanation coz counting of cells seem mandatory in cell cycle experiments....as in my first experiment I didn't do that, from my second experiment I counted the cells with a haemocytometer in both the cases (Negative Control & Treated) and repeated it twice. The shift unfortunately exists.....

and Dr. Lange thanks for sharing this, as I didn't actually know about this phenomenon of complete removal of histones from a DNA @ higher PI concentrations.

In addition to that I'd like to mention that, my extracts are showing a significant Cell Cycle delay at that dose concentration.

Hi Abhishek, then it might be a real effect of your cells itself as Barry Barcley suggested. Why do you not go with different concentrations of your treatment to see if the shift is obvious to change?

which plants extracts and which concentration you have experimented. i can do the same and justified.

@ Nicole Foster: I will definitely try that. The concentrations m using now (especially for flow) is just below the LD 50 for mouse and IC 50 for U87 (different in both).

When although I am increasing the dose to double i.e. 80 micrograms per ml in HPBLs in case of cell cycle kinetics (done via classical FPG technique) is not showing that significant delay which was otherwise shown by the lesser dose.

I thats why ignored that. Although @ that particular dose the extract protects the cell cycle kinetics when I analyse their ability on Radioprotection in HPBLs (chromosomal aberration study); where this is an important concentration!

I was thinking of one weird Idea though....I will just simply isolate the respective DNAs from both the samples and look for its DNA content in nano drop.

You can try that (even I never heard of people doing it) but you have to make sure that you will start with the same amount of cells even you can not be sure that you will loose the same amount in both samples. You can also try to normalize you nuclear DNA to your mitochondrial DNA for example as an internal control (to get ride of the effect of loosing sample during the purification) by using a gene encoded by the nuclear DNA in comparison to a gene encoded by the mitochondria. What if you are doing immunofluorescence of your samples with DAPI and a mitotic marker to see the structure of the DNA in the cells? I am not quite sure if it will work.

Sounds strange!!

But issue can be sorted out for sure, make it more clear,

1. did u use RNase?

2. What are the differences you are seeing in the Scatters?

3. if you could share the FCS files that will be great more infos can be revealed.

4. can u also send the protocol wht u used to prepare the sample?

Senthil.

Dear Senthil Nathan,

Thanks a lot for your concern

Yes I had used RNase. The incubation step was @37 degree celcius for 1 hour for every sample.

In the Scatters I had observed that the primary mob of cells shift upwards linearly, that is show more forward and side scatters. Say what happens when you increase the voltage.

Although all the treatments are taken at the same calibrated values done with control.

Protocol in brief was:

1. Fixing the Cells with 70 % chilled et-OH (kept thereafter overnight @ 0 degrees)

2. centrifuged, discarded the supernatent

3. Rehydrated with PBS (1X)

4. Incubated with 50 microlitrs of 50 micrograms/ml RNase @ 37 degrees for 1 hour.

5. Added 200 microlitrs PI working Soln, incubated for 5-6 minutes at RT

6. Acquired in flow.

Dear Nicole Foster,

I was wrong with quantification of DNA as you told and which happens to be the fact that we can't be sure of the exact amount of DNA of the cells and the actual yielded in practical.

Dear Abi,

This really grinds the brain, ok.

Lets get into the business if you treat the cells with RNase then it's perfectly obvious that you are looking only on the DNA.

1. The cells are behaving strange in the scatter you said, is it increasing more in SSC and Less in FSC?

2. I could belive if I were you, the extract which you are using could increase the cell's autofluorescence by some way. (But to confirm that you are really in the position to see them under any imaging system if possible do a DNA quantitaion too, which been mentioned in the thread somewhere, which is worth doing)

3. You can send the FCS files to my mail ID if it is not a problem for you, so tat I can analyse it and get back to you.

senthil.

I guess I can send you the FCS files, if I won't be bothering you.....thanks a lot or the help. Kindly pass me ur email ID

wow! what a great discussion. though, I am not an expert of FACS, I am sure approach of Senthil is appropriate. Best wishes to Abhishek.. and Thank you all .....

[email protected],

Download: www.ieType.com/f.php?Fzer7t

sorry for the delayed reply.

etype doesn't work in our campus server.

Then, You send me the FCS files to my mail, I reply to your mail.

@ Senthil

Kindly pass me your email ID. Its not there in your profile.

I will send the FCS files rightaway.

Thank you

@ Pavan

Yes you are very right. The extracts at times mess up the readings (in my other non-flowcytometric experiments). But to the best of my knowledge I guess PI would not prefer to stain any other non-nucleic acid content in the samples, including the debris or particulate part of the leftover extract.

Even one of my extracts (methanolic) when is dissolved in water/PBS for treatments doesn't seem to clearly dissolve.

[email protected]

Dear Abhishek,

I agree with Pavan, it might be the extract which is producing an auto fluorescence. As i see there is no other reason for the shift in FL2. You should use negative control which has only cells and treatment of extract, but no PI and compare this with control cells with no treatment and no PI. if you find the difference in the shift in peak in your treatment, you can conclude it is your extract which is giving auto fluorescence in FL2. In that case you can take FL3 channel , as PI has broad range of emission and if unfortunately there also you get the same problem, then you can not use PI for such experiment. In that case You can use DAPI or Hoeschst for cell cycle analysis using FL4 channel(UV channel).

Try this you may get your problem be solved

All the best..................