I am planning to sort T cells from mouse lymph nodes. The downstream application of sorted cells will be adoptive transfer to mice. I am thinking about using PI (propidium iodide) as a viability marker for cell sorting. But I've heard that PI per se is toxic to the cells. Do you think using PI will be fine considering downstream adoptive transfer? Cells will be stained with PI right before the sorting. As you know, there is no washing process after PI staining so that presort cells will be exposed to PI for a long time. Or should I use amine-binding dyes such as live/dead aqua?
Hello Kyungho Lee
Viability dyes such as propidium iodide (PI), DAPI, 7-AAD will only enter cells whose membranes have been compromised. Once they do, they will bind to DNA and fluoresce so that these cells can easily be detected and gated out of any analysis.
On the other hand, protein-binding dyes also known as amine-reactive dyes work on the same principle that dead cells have compromised membranes but work in a slightly different way. Instead of binding to DNA they bind proteins so if they are added to a sample, live cells will bind some dye as there will be proteins present on the surface of the live cell, but dead cells will bind much more as the dye can enter the cell and have access to the abundance of intracellular proteins.
I would suggest the use of DNA binding dyes like PI or 7-AAD. 7-AAD can penetrate cells over time if not washed off, thereby reducing the pool of live cells available to sort. Because of this, viability dyes like 7-AAD and propidium iodide should be added just before running your sample.
The toxicity effect of PI will depend on the cell type. The incubation time with PI and the buffer in which the cells are suspended also play an important role. During incubation with PI, use 0.1% BSA in 1X PBS that will help protect the plasma membrane. You should use amine dyes preferably for fixed cells.
Best.
PI is not good cause gives you trouble in compensation too. definitely better aqua live/dead or similar, you can add aqua dye while staining for your surfaces markers, so no extra work. good luck
7-AAD can be a good dye for this applicaiton. You need to make a validation study before you apply it to your research.
Gulderen Yanikkaya Demirel HI Dulderen, is there any advantage for using 7-AAD over PI?
hi, just remember that 7AAD is not stable link and it is not fixable if you have to do intracellular staining in combination with live?dead 7AAD is not ok
Michela
Kyungho Lee for successful staining with PI you need to use RNase, but in our hands 7-AAD works well and is more stable compared to PI.
I am writing down an article that has compared PI and 7-AAD for sperm viability.
N Falzone et al. Comparison between propidium iodide and 7-amino-actinomycin-D for viability assessment during flow cytometric analyses of the human sperm acrosome. Andrologia 2010 Feb;42(1):20-6. DOI: 10.1111/j.1439-0272.2009.00949.x
7-AAD is less bright in comparison to PI, but staining is stable.
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