Hi, everyone:
Recently I perform Intracellular cytokine staining (ICS), and meanwhile I want to identify live cells by propidium iodide (PI) staining. It's necessary to fix and permeate cells for intracellular staining, so all of the cells are permeable, and which is difficult to identify live cells by propidium iodide.
Does anyone have any solutions? or is there other approaches to identify live cells and stain intracellular cytokines at same time?
Since FSC/SSC pattern will be changed after cells are performed ICS, so it's essential to do the ICS procedures even I just perform PI staining.
Hope someone could provide me good solutions. Thanks!
Don't you stain with PI before the fixation/permeabilization? If you do that, then wash away the PI and then fix/permeabilize, then ideally, no new PI should stain the permeable cells.
Hi Kuan, You should switch from PI to one of the fixable viability dyes. They are called Live/Dead fixable viability stains at Thermo Fisher and Zombie dyes at BioLegend. There are also a lot more color options available to use with the fixable dyes.
you need to use 7aad, its a better marker for cell death than PI, has better emission at 488 nm, and can be used on cells that have been permeabilized using a reversible detergent like saponin. PI will not work for this.
Please see page 5.3.11 from Current protocols in immunology.
https://www2.vet.cornell.edu/sites/default/files/PrepReagents.pdf
Dear Shen-An Hwang ,
Thank you for your answer! Indeed I‘ve done that staining with PI before the fixation/permeabilization, however it’s surprisingly that live cells were PI-positive, which was unreasonable. Live cells supposed to be PI-negative.
I’m not sure if there are any wrong procedures I’ve done or the wrong analysis.
I will try it again!
Hello,
As stated by Nicole Acuff, amine-reactive dyes are the recommended reagents in this ICS setting. They are available at many suppliers (eg Miltenyi, BD, etc...). They are very bright, available at different fluorescence channels and entirely fixable. You really should go with them to avoid annoying remarks from reviewers!
Hello!
Use fixable-viability stains. The method is almost the same, but you won`t get troubles. Biolegend has nice Zombie live/dead stains.
Yes you can do that. Permeablization step is not permanent. Usually permeablization occur for the time agent is added (in most protocols, antibody staining is performed together with perm Buffer incubation or in the perm/wash buffer), so that the reagent make the membrane pores bigger to allow the antibody to get in. and then the pores are normalized due to elasticity of membrane.
Even if you add PI after the intracellular staining, it would not create unmanagable mess. But if you add PI initially (before perm step), it would also be great.
As PI has broad spectrum of emission it may cause spill over in your other fluorescence chennal. Try to use some other DNA dye with bright colors (if you have brilliant violet options available in your cytometer).
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