Fri Mar 25, 2022 5:05 pm by GregBehbehani
Hi Simon,
I can agree with what has been said by everyone else so far. If you are primarily staining intracellular antigens, the partial-perm barcoding buffer (which is what I think Fluidigm provides with the kit) is unlikely to be good enough to get good staining of the intracellular antigens, and that may be the issue that your are having. The protocol that I published was specifically designed to NOT fully permeabilize the cells, as we would almost always do a methanol perm step before doing the intracellular stain (as Peter Krutzik had long before shown methanol to be a better perm for the things we routinely looked at). We also saw that a full saponin perm could actually strip of some surface antigens (though this was pretty rare). Though it is worth noting that fixed cells (particularly if they were fixed a while ago, or fixed and then frozen) have typically lost enough membrane integrity that the barcoding will work without the additional perm, but it requires more barcoding reagent, and was less consistent in my hands. Also, if you have some cells that were dead at the time fixation, they typically have a membrane that is more permeable and end up with brighter (sometimes too bright) staining. Adding the 0.02% saponin was the minimum amount that consistently eliminated all of this variation, but also did not really allow for full staining of intracellular antigens. What we did not appreciate at the time was how incredibly variable different saponin lots are. Saponin is not in any way a pure substance, and there is very little standardization of the available products. Additionally, some saponin products cause non-specific staining with Fluidigm's newer labeling polymers (which was the source of a product recall several years ago). Fluidigm does now test their saponin lots to make sure that this doesn't happen, but they still don't give very much detail on what exactly is in their buffers, or which polymer is on which antibody that they sell.
I'm not contractually permitted to discuss Fluidigm's barcoding kit, but I can say that I think Zach made some excellent comments on this.
Since you don't need surface marker staining, you may just want to fully permeabilize your cells before barcoding, with whatever perm protocol works best for the antigens you are interested in. Then when you barcode, you can get by with much lower amounts of Pd reagent, and can get 2-3x more uses out of each barcode kit. Like Zach, we also bought our own isotopically purified Pd. If you skip 102, this isn't terribly expensive for 10-population barcoding, but we went ahead and purchased all 6. The cost is pretty high upfront, but you will basically have a lifetime supply of Pd.
Good luck,
Greg