Intracellular cytokine staining can be challenging to get right. The cells need to be in great condition to withstand the stimulation without large losses in viability. The stimulation also needs to be optimized to get the maximal signal for whatever cytokines you're staining for. The stimulation and protein transport inhibitors will affect the biology of the cell, so markers may not retain the expression patterns you're used to, or the proteins may actually be inside the cell. Fixation is also critically important; the wrong fixative will cause the cytokines to be released from the cell, negating all that hard work.
Let's look today at the role of ionomycin in this process.
Ionomycin is the most commonly used calcium ionophore for intracellular cytokine staining. It activates calcium flux in cells, mimicking what happens with strong antigen receptor signalling. Combine this with phorbol ester (PMA) stimulation to activate protein kinase C and NFkappaB, and you've got a strong polyclonal stimulus that will activate most cells.
Clearly, this isn't what you want to use if you're interested in measuring antigen-specific responses. It does, however, allow you get a broad snapshot of the immune response across a range of cell types, for instance T, B, NK and myeloid cells simultaneously.
It's important to get the concentration of ionomycin right. Ionomycin from different suppliers or even in different lots can vary in potency considerably. For best results, titrate the ionomycin in a stimulation. You'll also want to prepare single-use aliquots and store them frozen, preferably at -70C.
In a good lot, the optimum will be between 500 and 1000 ng/ml (in other words, approximately 1uM). Too little, and you won't stimulate the cells to produce all they're capable of. Too much and the cells will start dying off, some more than others, biasing the results.
In this example, we've got CD3+ viable T cells from either human PBMCs (bottom) or mouse combined spleen and mesenteric lymph nodes (top). In each condition, the cells are also treated with 500 ng/ml PdBU and 1000 ng/ml brefeldin A.
More tips here.
Some relevant resources:
Lamoreaux, Nature Protocols 2006. This paper covers a lot of the fine details intracellular cytokine staining. It's a very good starting point.
Lovelace and Maecker, 2014. Many tips at the end.
An overview from BD.
A basic protocol from BioLegend.
Stimulation recommendations from BioLegend.
Stimulation recommendations from ThermoFisher.
Comments