I treated my cells and tested the cell cycle distribution using PI staining and flow cytometer analyses and I saw a dramatic increase in subG1 phase. I then proceeded with Annexin V - PI staining, but there was no effect (I did see effects with other treatments and the positive control so it's not a matter of poor staining or flow settings).
I can't think of any scenario that would give these sort of results. Anybody have an idea?
There is a good chance your SubG1 cells undergo blebbishield emergency program. Collect the floating cells (obviously the population causing subG1) after treatment along with medium, spin it down at 1200rpm for 3mins and discard the supernatant and resuspend gently (using a wide mouth pipette; with minimal pipetting) in fresh complete medium and plate the cells in new plate/flasks to see whether it forms spheres or reattaches without forming spheres. Alternatively, if you have FACS sorting facility, sort the cells from SubG1 and wash+plate as described above. For more details on blebbishield emergency program see my paper
https://www.researchgate.net/publication/233742776_Blebbishields_the_Emergency_Program_for_Cancer_Stem_Cells_Sphere_Formation_and_Tumorigenesis_after_Apoptosis
Good luck..!!
Article Blebbishields, the Emergency Program for Cancer Stem Cells: ...
might be the limitation of Annexin V assay or procedure of staining...
Dear Emily, Yes your results are surprizing given you have SubG1 positive samples. Just a few questions about the data you have - are there a lot of dead cells PI+AnnV+events and not just AnnV+ve - this would indicate that you would need to do the Annexin V assay at an earlier time point. Another explanation would be that you have late apoptosis only in the samples that is what the SubG1 peak is indicating and no early apoptosis. Maybe it is this particular treatment or the time you are sampling these cells that they are missing this stage of cell death - given you say you have other treatments that have worked - maybe a fuller explanation of your results would be helpful - hope this helps Gary
The presence of sub-G1 could depend on a setting wrong of cytometer(e.g. you have considered the debris) on the other hand the essay ANX V / PI is more sensitive than the information the apoptosis extrapolate on cell cycle analysis. I believe the results obtained with anx V / PI and I would do a morphological analysis under the microscope, to assess the actual presence of apoptosis.
Hi Emily,
if your subG1 "population" is clearly separated from G1 in the PI-Histogram, then you might have fragmented cells, nuclei and whatever. If your subG1 is "fused" to the G1 peak I would consider this as real apoptotic cells. Only in the second case you might get annexin V staining.
cheers
Michael
In your PI stain, did you add any surfactant or detergent, i.e. saponin? Even a slight amount of cell membrane shearing during the procedure may release nucleic acids and cause a "sticky" aggregation of cell pieces, which may appear to be "sub" G1. Just a thought.
I agree with Anna. Look at your cells with the light microscope to identify any morphological alteration or contamination and with the fluorescence microscope to determine the presence of apoptotic cells. Furthermore a too prolonged exposure to the drugs could be the reason of your finding: so reduce incubation times.
Many of the assumptions of the previous responses can be true.
The method I published in JIM (1991) for the flow cytometer analysis of apoptosis works very well on thymocytes and cells in suspension but is less accurate on some adherent cell lines. In these, debris and detached cells can contribute to subG1-peak.
In these cases a careful microscopic analys of the sample is mandatory.
Bye, -- Ildo
Hello Emily: Some, if not all, of the kits used to prepare cells for cell cycle analysis contain detergent to permeabilize them, thus removing the plasma membrane. After applying the cell cycle protocol instead of cells you should have nuclei. When you are processing the cells (nuclei) for cell cycle first, most probably by the time that you perform apoptosis/necrosis assay, you do not have the phosphatydylserine anymore, necessary to bind to annexin V-FITC. Therefore, you are not able to identify cells with green fluorescence signal. All your cells should appear as necrotic cells, emitting the propidium iodide signal, without any viable cells. If you are detecting some viable cell population (events without green or red fluorescence signal) they should be cell fragments without nucleic acids inside. Hence, it should be convenient to use separate aliquots of cells for each individual assay. Hope this help. Good luck! AV
Hi Emily,
I agree with Michael and Gary, If you have a fused peak, you would consider this as late apoptotic cells. But if you have two separated peak, you can consider this as early apoptosis, Only in the second case you might get annexin V staining.
Hi Emily,
Could you post your PI peaks+gate settings to confirm SubG1? Of course you can keep cell name drug name etc. confidential. Just needed the peaks+PI intensity axis+cell number axis.
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