Hi Anshuli:

It is a bit weird, from your back gating, there are some debris stained, so there are 3 works i can think of:

(1) Check staining protocol an get rid of debris to see whether the middle peak is still there; avoid any technique mistake.

(2) Try Edu+Hoechst to stain cells to split cell into G1, S and G2/M phase clearly to see whether there are homogenous S phase cells;

(3) Giemsa stain cells and look chromosome under microscope;

If you still find some cells arrested in S phase, big congrats, you got something big.

Hope this helpful.

Regards

Hi Shiqiu,

Thanks for the suggestions. Edu + Hoechst staining is something that we can easily do and see what's happening (someone had suggested us to do Ki-67 staining). We were thinking of having the cells karyotyped as well.

Coming to the gating strategy, we tried to locate the cells in middle peak (and to rule them out as doublets).If you see the image, it is the population in green and definitely not doublets.

If you have any other suggestion, please feel free to post it here!

Many thanks.

Kind regards,

Anshuli

Hi Anshuli:

Thanks for clarification, yes, the middle peak are not debris.

I am not so sure the benefit of Ki67 staining, since almost all proliferating cells express this protein. Edu + Hoechst staining can validate your observation and avoid any potential tech problems.

Since the PI staining showed some homogeneous DNA instead of evenly expression amount, (1) it may come from strange sub-population (2) some S phase arrest induced synchronization. In this case, look chromosome structure by Giemsa staining or Confocal look DAPI stained DNA may give you some clue.

Regards

Shiqiu

Hi Shiqiu,

Thanks for the suggestions, we are going to try the Edu + hoechst staining for our cells to get some more details. However, I am not sure how DAPI staining by confocal is going to help? Wouldn't it stain the whole of nucleus but not the individual chromosomes?

Also, in your experience,do you know of any cancer cell line that has heterogenous population? I tried to look up online, but couldn't find anything relevant.

Thanks.

Regards,

Anshuli

Hi Anshuli,

It looks like you have may have a population of cells that are stalling in S phase. You can do a pulse/chase BrdU or Edu experiment and look at progression through S phase over time. Sometimes when cells are very genomically unstable they have difficulty progressing through S phase.

Good luck!

Hi Anshuli:

To be honest, i don't know what you can see under Confocal to look DAPI stained cells. But, if there is a homogeneous S phase cells, the whole nucleotide pattern should be different from normal cells. Combining chromosome image, you may have a chance to get some clue if you are lucky enough.

Regards

Shiqiu

Hi Janice M Pluth,

Thanks for replying! Glad that you mentioned about the genetically unstable cells being arrested in S-phase; I remember seeing an article about unstable cancer cells (taken from patients) having heterogenous populations (and same weird middle peak) being linked to resistance to drugs.

We are going to do the Eud + Hoechst flow cytometry in coming weeks, but I am not really sure what to expect. One concern I have is that if the middle peak is actually S-phase cells, the software (FlowJo or others) will probably have hard recognising it which means I won't be able to quantify?

Kind regards,

Anshuli

Hi Anshuli,

Very interesting issue, Ive been doing Flow with PI for my work for the last 3 years and its very interesting to see a clear peak between G1 and G2.

Few questions: is it your control group or treatment ?

also can you plot PE-H or PE-W vs PE-A and send the pic again ?

Are you using any kit for staining or PI with own protocol ?

If there is an option to sort those cells, then you can stain or do qPCR for ki67 and p21 to see if the cells have stalled in S phase. Some times you might see ki67 high (indicating cells are progressing through cell cycle) but might have high p21 as well (inhibitor for cell cycle progression) in that scenario, the cells do tend to halt in certain stage. If its your treatment group then its worth looking at, if your treatment is disrupting the checkpoint of progression to G2.

I would also repeat this with different timepoint cells. Just to make sure that cells are progressing and when they reach S phase, they halt.

Hope this helps.

Kiran.