it sounds to me as a staining issue. I think you need to give us more details of your staining protocol.

Thank you for your time Dr. Avitabile. After washing the cells with ice cold FACS buffer, I resuspend them in 50 ul of binding buffer and add 5 ul of Annexin V-FITC  per 100 ul of cells suspension in binding buffer. leave them for 10-15 minutes and then ad the same amount of PI. Then I add the cells to a FACS tube containing approximately 200 ul of FACS buffer and then I subject the cells  to a flow cytometer.

I`ll explain if more details are required.

No actually, I think the is correct. Do you stain mononucleate cells right?

Any difference in the number of cells do you use as control in comparison to your treatment? Is it possibile that you have any chelator of calcium in your treatment? In that case anV binding should be impaired so that you are costrict to rise the voltage. When you go back to control you have an issue with compensazione since the detection of green channel is too High. Otherwise honesty I have no clues right now.

Different treatments may change the levels of autofluorescence of the cells. If this is the case, we can't use the same bar for the control sample and the treatment groups to determine pos/neg.  For each treatment group as well as the control sample, you may keep a fraction of cells as unstained to determine this bar. That means the unstained cells should receive the same treatment (for each treatment group and untreated group) and should be submitted to the staining protocol but without adding Annexin and PI.

Are you gating FSC and SSC?

If you are using Sigma kit try 2.5ul of Annexin and 1ul of PI, this usually gives optimal fluorescence intensity to compensate FL1/FL2

Dear Dr. Avitabile,

I used to isolate peripheral blood mononuclear cells from whole blood collected in EDTA-CBC tubes and then do my treatment and staining whithout any Calciuk chelator. Do you think that isolating PBMCs from heparinized blood might interefere with the later stages of staining isolated and washed PBMCs??

I really appreciate your valuable guide in advance.

Dear Dr. Gu-Trantien, 

That sounds to be a really interesting method to have more percise controls. Comparing treatments with such a control can give better results considering the possible effect of our treatment on the auto-fluoroscecne  of the cells. I will try that for sure. Thank you

Ann Atzberger 

Yes! Actually this is one of my problems also!

In the SSC/FSC plot for all PBMCs that I have isolated so far I normally see two completely distinct populations (the attached file- from left to right: unstain, control, treatment). What can these two populations be? Do they display alive and dead cells?

I have stained them with CD45 and I am sure that none of these two populations are debris and they are true cells.

Nina Porakishvili

Thank you for your answer, but I am using BD.

I would recommend Sigma kit, it works very well in this proportion. Never used BD, but even for the Sigma kit my students tried different volumes and proportions before reaching this optimal one, and this is not the one recommended by the manufacturer.

I would therefore suggest that you might wish to try different combinations of volumes for Ann or PI. Try to decrease the volume of the reagent instead of taking the voltage down.

The large population in the right-hand image looks like dead cells, in fact dead cells seem to prevail in all three. I would use log for SSC.

Sorry Milad but it seems that there're almost no viable cells in your treated cells. The FSC/SSC population on the right/low side corresponds to the viable cell gate.  If your control cells received the same treatment, then this may be due to the staining protocol ? You left them too long time ? I didn't do a lot of apoptosis experiments but I know for this staining, we need to lecture within 15min following staining.

Dr. Gu-Trantien,

So you say that the population on the right/low side correspond the viable cells? if so, that is a perfect and desirable result for me!!! Because I`m treating the cells to induce apoptosis and consequently cell death (the cells are malignant cells) (I am evaluating the apoptosis-inducing potential of the substance)! 

But my question is that why do I have that many dead cells in my control which is not treated??!! (my control contains untreated cells stained with Annexin-V and PI). 

Should I continuously work on ice?

Actually, my staining protocol takes much longer than 15 minutes. Is that the reaon for so many dead cells?

I really appreciate your help and I find your answers so useful.

Regards

Dr. Nina Porakishvili

I will test different proportions to achieve the optimal staining protocol. Yes, I got so many dead cells in my treatment and that is kind of desirable because I am about to induce apoptosis. However, Most of the cells has absorbed PI in the treatment group but not Annexi-V!! What does that mean? Does it mean that my treatment is inducing toxic effects which leads to the death of cells directly without undergoing apoptosis process? 

Is it possible that cells in the late stages of apoptosis be only positive for PI (Like what I got here!) or should they be positive for both Annexin-V and PI??

Milad, If you are working on tumor cells extracted from fresh tissues (not cell lines), it's normal for me that many of them die without treatment. I'm working on tumor-infiltrating lymphocytes, which are much easier than cancer cells because they are much more resistant (with much higher viabilities) than any other cells. For the duration of the staining protocol, I mean when the staining is finished, you need to acquire samples within 15min, as soon as possible. I see no differences between your unstained and your control samples so there should have no problem for your staining protocol. Maybe try to get more viable cells in your treated samples by decreasing the duration or the doses of your treatments ? If you need to treat samples for several days, maybe you can try to remove the dead cells before treatment (Myltenyi has kits easy to use but with a risk of cell loss). If you treat your cells only for hours, you should try fewer hours and lower concentrations of your reagents.

Dr. Chunyan,

That was interesting. Yes, I am working on leukemic cells isolated from the blood samples of patients with different types of leukemias. Sometimes I isolate only CD34+ cells using MACS and do the treatment.

I also think that leukemic cells normally have a high number of dead cells due to their instability and also I have experienced that the yield of PBMCs from the blood of leukemic patients is dramatically less than that of normal individuals.

Anyway, you helped my so much and I can now made sure that there is no significant problem with my protocol and having a number of dead cells is normal when working with malignant cells.

It would be a great pleasure to stay in touch regarding our research since we are both working on cancer and it is my primary interest.

I would be careful about the above conclusions. How long after you receive the sample are you staining for Apoptosis? Are the PBMCs frozen beforehand? If yes is freezing before or after Macs. Are you always enriching for a population of interest?

Dr. Atzberger,

I do not freeze the cells. After I obtain whole blood from leukemic patients, I immediately isolate PBMCs using ficoll and after washing and counting the cells, I distribute them into different wells (unstain, untreated control, and different treatments). After 24, 48, and 72h I stain the cells with PI and Annexin to evaluate apoptosis. I normally do not focus on a specific pooulation and I rvaluate PBMCs as a whole.

I presume this is because you suggest that the majority of the cells you isolate are leukaemic? It would be still useful to use a pan-marker to delineate a leukaemic population.

My group works on CLL, and the level of heterogeneity of spontaneous apoptosis is high. I would worry that all cells are PI+ without going through the double-positive stage first, particularly after 24h.

It could well be that you observe a toxic effect, rather than apoptosis.

Why not to stain for Ann+PI+  the cells straight ex vivo, then after shorter periods of time post-treatment with your drug, such as 1h, 2h, 4h. You should see no apoptosis as early as 1-2h post-treatment, so if the cells are dead, it would indicate toxicity. Your untreated controls should be the same as ex vivo. Likewise, if you use leukaemic marker, and your drug is specific for leukaemic cells you should be able to see if all PBMCs are dead (toxicity), or the leukaemic cells exclusively.

Dr. Porakishvili

Absolutely useful comments, I will consider them for sure. 

Thank you for your valuable help

Good luck with your research!