Quantification of T Cell Antigen-specific Memory Responses in Rhesus Macaques, Using Cytokine Flow Cytometry (CFC, also Known as ICS and ICCS): Analysis of Flow Data (2014)

What was initially termed 'CFC' (Cytokine Flow Cytometry) is now more commonly known as 'ICS' (Intra Cellular Staining), or less commonly as 'ICCS' (Intra Cellular Cytokine Staining). The key innovations were use of an effective permeant (allowing intracellular staining), and a reagent to disrupt secretion (trapping cytokines, thereby enabling accumulation of detectable intracellular signal). Because not all researchers who use the technique are interested in cytokines, the 'ICS' term has gained favor, though 'CFC' will be used here. CFC is a test of cell function, exposing lymphocytes to antigen in culture, then measuring any cytokine responses elicited. Test cultures are processed so as to stain cells with monoclonal antibodies tagged with fluorescent markers, and to chemically fix the cells and decontaminate the samples, using paraformaldehyde. CFC provides the powers of flow cytometry, which includes bulk sampling and multi-parametric cross-correlation, to the analysis of antigen-specific memory responses. A researcher using CFC is able to phenotypically characterize cells cultured with test antigen, and for phenotypic subsets (e.g. CD4<sup>+</sup> or CD8<sup>+</sup> T cells) determine the % frequency producing cytokine above background level. In contrast to ELISPOT and Luminex methods, CFC can correlate production of multiple cytokines from particular, phenotypically-characterized cells. The CFC assay is useful for detecting that an individual has had an antigen exposure (as in population screenings), or for following the emergence and persistence of antigen memories (as in studies of vaccination, infections, or pathogenesis). In addition to quantifying the % frequency of antigen-responding cells, mean fluorescence intensity can be used to assess how much of a cytokine is generated within responding cells. With the technological advance of flow cytometry, a current user of CFC often has access to 11 fluorescent channels (or even 18), making it possible to either highly-characterize the phenotypes of antigen-responding cells, or else simultaneously quantify the responses according to many cytokines or activation markers. Powerful software like FlowJo (TreeStar) and SPICE (NIAID) can be used to analyse the data, and to do sophisticated multivariate analysis of cytokine responses. The method described here is customized for cells from Rhesus macaque monkeys, and the extensive annotating notes represent a decade of accumulated technical experience. The same scheme is readily applicable to other mammalian cells (e.g. human or mouse), though the exact antibody clones will differ according to host system. The basic method described here incubates 1 × 10<sup>6</sup> Lymphocytes in 1 ml tube culture with antigen and co-stimulatory antibodies in the presence of Brefeldin A, prior to staining and fixation. <i>Note: This is the second part of a two-part procedure. Part one has the same initial title, but the subtitle "</i>From Assay Set-up to Data Acquisition <i>(</i>Sylwester et al., 2014<i>)". The Abstract and Historical Background is the same for both documents</i>.