A short essay on an immunological technique
Cytokine production by flow cytometry (intracellular cytokine staining)
Cytokines are communication molecules which are secreted by particular cells of the immune system and which intervene in the regulation of immunity, haematopoiesis and inflammation. Flow cytometry is a powerful technique for analysing individual particles, molecules or cells. It involves the passing of numerous cells (a few thousand) per second through a laser beam and capturing the light that emerges from each cell as it passes through.
Intracellular cytokine staining is a popular flow cytometry based assay which detects the frequency and type of cytokine produced. It permits synchronized characterization of cytokine synthesis and surface proteins. The researcher can gain significant facts about the reactive population and even the category of immune response obtained.
In the assay, the cell population is isolated and the cells are activated in the presence or absence of a stimulus. To prevent cytokines from being secreted, the use of an intracellular protein transport inhibitor such as “GolgiStop” is necessary in the last 4-6 hours of cell cultivation. This results in the build up of numerous cytokine proteins in the Golgi complex. This in turn leads to an improved facility of detecting cytokine-producing cells. The next step is to stain the cell surface marker, which, as the name suggests, is located on the cell surface and determines a particular cell type. Cell fixation is then applied with the help of paraformaldehyde which preserves cell morphology. The cells then go through permeabilisation. This allows penetration of the cell by the the fluorescent cytokine-specific monoclonal antibody which is added. Finally, the cells go through a washing and fixing process. The cells are ready for flow cytometry analysis. In optimal flow cytometric analysis, the stained cells are examined by light microscopy to verify their dispersal. Upon data analysis, dot plots can be produced; they demonstrate the rate of occurrence and pattern by which particular cells express certain amounts of intracellular cytokine proteins and cell surface antigen.
Advantages and disadvantages
Intracellular cytokine staining does not rely on the recognition of secreted cytokines but on the evaluation of particular cells within a population. As seen previously, this technique is used in parallel with surface staining to permit assessment of the particular cells which secrete cytokines. Cells which present comparable cell surface morphology but which produce different cytokines and have different functional features can be studied with this technique. Cytokine production by flow cytometry provides in depth information about individual cells. It is frequently used for homogenous populations, since it is certain to expose heterogeneity, if this arises, and is accurate in removing fragments or dead cells from the ultimate values.
In contrast, intracellular cytokine staining tends to overwhelm with exhaustive information which may be useless. Another disadvantage comes from the purification process. For this to be reliable, even on a high pace purifier, the cell flow speed cannot surpass the couple thousand cells per second. For example, when a couple cells pass through the laser too close to each other for it to make a distinction, the protocol is that they must both be spared. Flow cytometry passes, on one hand thousands of cells per second, which seems like an elevated number; on the other hand, most experiments would need many more cells. The cost of operating the cell purifier is high, and another problem might be encountered: in order to obtain optimal results, the cells need to be kept in the best conditions and this becomes tricky when the process is slow. A final problem may arise considering sterility. In order to keep the environment clean, the cytometer has to run ten times slower, again elevating the cost, which is at a few hundred pounds per operation.
ELISA (Enzyme-linked immunosorbent assay) or ELISPOT (Enzyme-linked immunosorbent spot) are alternative ways to measure cytokine production, the latter being developed from modified version of the former. They allow the recognition and listing of cytokine-producing cells and permit the observation of the secretary product of particular activated cells. In the assay, a spot corresponds to a responding cell. These methods are rapid and simple; the cost of executing them is much less and they permit a more important number of samples to be processed per day.
Example of technique used
Duramad et al (2006) in Environmental Health Perspectives Volume 114, Number 12 investigated chemicals to which children in rural settings are exposed to, who in turn trigger a “maturation of the immune response in early childhood”. The study involved intracellular Th1/Th2 cytokine analysis by flow cytometry. The CD4+ population was studied for expression of IL-4 and IFN-gamma. Th1 cells corresponded to cells that stained for IFN-gamma (bottom right quadrant) whereas Th2 cells corresponded to the cells that stained positive for IL-4 (upper left quadrant). Further statistical analysis revealed that the elevated Th2 status in early age children is in correlation with asthma and wheeze apparition in 24 month old children.
Brian E. Crucian, Raymond H. Widen (1997), The Use of Flow Cytometry to Detect Intracellular Cytokine Production in Individual Cells
Jung T et al, (1993), Detection of intracellular cytokines by flow cytometry, 159(1-2):197-207
Journal of Immunological Methods Volume 243, Issues 1-2, (2000), Pages 107-124
Peter wood, Understanding immunology, Pages 24-26