As the study detecting that the cytokines using the intracellular flow cytometry; As the cytokines play a critical role as mediators of immune responses; and are generating by a variety of cells. Methods that can be using to measure cytokine levels in a sample and the differentiate between different types are therefore essential tools for understanding the human immune system.
As study developing techniques to measure cytokine levels. However, all of these assays are based on the assumption that all cells of a given phenotype produce similar levels and types of cytokines. This often leads to ambiguous results and makes it difficult to determine the presence of contaminating cells in the culture. It was for these reasons that there was an extensive search for a method that could identify single cells by using their cell surface markers and cytokine production profiles.
Mediators of immune responses
But identifying that the cytokines present inside a cell usually presents a problem due to weak intracellular signal. However, a 1993 study tried to circumvent these issues through a novel method that is now widely using for the detection of cytokines IL-1α, IL-6, IL-8, and TNF-α. Monensin is a carboxylic ionophore that interrupts the intracellular transport process. It binding to the Na+, K+, and H+ ions; disrupting the ion gradients in biological membranes.
This perturbs the Golgi complex and transport to the cell membrane without interrupting the protein synthesis, subsequently leading to the accumulation of cytokines in the Golgi complex and signal-to-noise ratio increase. Human peripheral blood mononuclear cells were activating with phorbol myristate acetate (PMA) and ionomycin. This was done in the absence of monensin in controls and the presence of monensin in test cells.
Combination of paraformaldehyde
But the study showing that different cells; such as human naive or memory cells secrete different kinds of cytokines. The cells were initially fixing using a combination of paraformaldehyde (PFA) and saponin; as these do not alter the scatter properties in contrast to Tween 20 and Triton-X. Using three-color FACS the researchers could find differences in the expression of IL-2, IFN-y; and IL-4 in memory and naïve T cells.
To validate the results, the researchers performed microscopy along with flow cytometry for ten samples and tested the levels of IL-2, IFN-y, and CD45. They finding that the microscopy results were highly correlating with the results from flow cytometry. The cells staining for various cytokines can be storing for 1-3 days in the dark. Thus, this method provides a means to characterize the cytokines in a heterogeneous population, screening cytokine patterns; and functionally characterizing the cytokine-producing cells.