Cytokine Analysis by Intracellular Staining

The advent of molecular techniques for the first time allowed for the specific biological characterization and a more clear understanding of the function of molecules synthesized by leukocytes termed cytokines. In addition, availability of the recombinant forms of these cytokines with defined sequences and in select cases molecular structure allowed for a rationale to group the growing list of cytokines and their cognate receptors. Cytokines in general are proteins or glycoproteins that are synthesized by a number of cell lineages, the best characterized are those synthesized by the hematopoietic cell lineages. Cytokines in general are either secreted, expressed on the cell membrane and/or held in a reservoir form within the cells that synthesize them. Several cytokines are synthesized in a biologically inactive form (procytokine) and need to be cleaved to release the active form, a notable example being tumor necrosis factor-α (TNF-α). Certain cytokines are held as storage depots within tissue areas within which their function is manifest. Thus cytokines that perform hematopoietic growth and differentiation appear to be stored as depots within stromal cell layers of the bone marrow, whereas those involved in tissue injury and repair are localized to the extracellular matrix, skin and bone and so on. Most, if not all, induce their biological effect by binding to their cognate receptors, which provide intracellular activation of signal transduction and second-messenger pathways. Most cytokines also appear to serve as growth factors or differentiation-inducing molecules for hematopoietic cell lineages.