Hi all
I study on a treatment for the cell lines, 4T1 and B16. For Flow Cytometric evaluation of apoptosis, I use the Annexin V-PI staining kit, But I always have a problem with negative control. Untreated cancer cells also have high early and late apoptosis rate!
For my experiment, I trypsinize the adherent cells for about 1 minutes (less or more) incubation at 37°C, Centrifuge (1500 rpm for 3 min), wash once with PBS, Centrifuge (1500 rpm for 3 min), add binding buffer, and finally stain cells with the Annexin V and PI.
Does anyone have any idea about this problem?
Thank you
Treatment of trypsin causes such damage to cells. There is litterature available for damage of cells after trypsin treatment due to its It's cationic properties.
Now that people are shifting to Accutase, which in better dissociation reagent and affect cell health minimally.
Even if you see tryphan blue staining after dissociation, you will see 80-85% viability only, That should correlate with annexin-V staining.
Treatment of trypsin causes such damage to cells. There is litterature available for damage of cells after trypsin treatment due to its It's cationic properties.
Now that people are shifting to Accutase, which in better dissociation reagent and affect cell health minimally.
Even if you see tryphan blue staining after dissociation, you will see 80-85% viability only, That should correlate with annexin-V staining.
You can try the following to get less percentage of AnnV/PI positive cells in negative cells,
1. Don't make your culture plate over confluent, not more than 80%.
2. You can also try by lowering the centrifugation speed, after trypsinization and gentle pipetting.
3. Double check the concentration of the dye and incubation time after adding the dye.
What is the percentage of untreated apoptotic cells?
As suggested by Pritam, lower the centrifugation speed to 300g, minimize the time in annexin binding buffer and try improving your culture conditions (see standard published protocols). Even trypsinized adherent cells should be >90% live when properly cultured. A relatively minor rate of apoptosis/cell death is expected and should not constitute a problem for your experiments.
Dear Pritam Sadhukhan, thank you for your reply. Actually, I've already tried these, but still got high rate of apoptosis.
I decreased the number of seeded cells, decreased the time of culture, decreased the amount of Trypsin and also its incubation time, decreased the incubation and amount of dye, but unfortunately still got apoptosis. May I use Cell scraper for cells collection instead of Trypsin?
Dear Gabriele Casirati, thank you for your reply.
Actually, the rate of apoptosis is very high, more than 80%. However, I checked my cells before trypsinized. The culture condition is really fine
Dear Raul Fernandez, thanks for your reply. Actually, I try to do all the process as fast as I can to prevent dryness of cells. However, I saw the high amount of apoptosis again and again.
As u mentioned, you have a experience in this, may this happen because of binding buffer? Can buffer cause to make high rate of apoptosis?
Can I use PBS instead of Binding buffer?
Hi Elena, you have to adjust the detachment condition. Trypsin is not ideal. May be try an anzymatic free buffer. Regards, Dmitri
1. Wash your cells with PBS with 2% FBS.
2. Pay attention to the temperature, too cold or too hot weather can effect on cells.
3. If you have to use trypsin, you should dilute your trypsin with PBS 2%. Because this reagent is not recommended for apoptosis essay with Annexin-V PI.
4. Incubation time with PI is so important. In commercial protocols, it is commonly suggested that you should add both Ab simultaneously. personally, I add PI later than annexin , For example 5 minutes before analyzing.
Good Luck 👍
Hi!
In my experience trypsin has relatively low effect on cell viability (after all, they still grow after being detached for splitting :)). Incubation in binding buffer does not impact the result heavily as well (I incubate for over 15 minutes, it's fine). What I see quite often is people very aggressively pipet cells up and down during collection and introduce air bubbles which kills cells. Centrifugation speed is also not something I would be worried about. Actually, for cell death analysis higher speed is recommended as it pellets smaller and lighter cell corpses as well (I use 450g, 3 min).
Another thing. How do you recognise apoptosis in your cells, if your negative control does not work? Perhaps the issue is not with the cell death, but the gating you're applying in your experiments. Would you be willing to share dot plots of your experiments? Maybe this would clear things out a bit?
Good luck!
Bartosz
Sometimes you see a shift in the negative population. You need to show the dot plot
Myself and my team have been consistently doing AnnV/Pi and DioC/PI staining on 4T1 and B16 cells and in control conditions they look really OK (normally
Dear Lorenzo Galluzzi, Thank you for sharing your experience here with us. I'm also using B16 and 4T1 cells in my work. The results from control of B16 is better than 4T1. 4T1 always gave me high apoptosis rate than B16. However, I've already ask another person to do this for me to delete any problem maybe from my hand, again saw same results
Hi Elham!
Did you observe a decrease in size in the apoptotic population? The ANX V positive particles can be apoptotic bodies or other extracellular vesicles, such as oncosomes (1-10 um), presenting PS on the outer membrane.
You can try using critic saline to harvest the cells. Citric saline is simple to prepare and is not harsh for the cells.
also you can reduce the concentration of PI
Hi dear Jan Balvan. Actually, I have not seen any decrease in size. I just observed the high rate of apoptosis in my control samples.
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