Manipulation Of Cell Populations Research Articles

Manipulating peripheral blood cell populations to mimic immune disease states: a novel approach to teach flow cytometry to undergraduate immunology students

Abstract Flow cytometry (FC) allows for the quantification of individual cell populations whose levels are known to change in disease states, such as, the reduction in CD4+ T cells in AIDS. While FC is a powerful technique, it can be difficult to teach its principles and applications to undergraduate students. Assessing changes in cell populations is an excellent approach to teach FC, however obtaining real patient samples is not often possible. In addition, hands on use of a flow cytometer by students is difficult or impossible to achieve when class sizes are large. To address these challenges, we taught the principles of FC, sample preparation (Week 1) and data analysis (Week 2) of mock “control” and “patient” samples. In Week 1, the theory of FC was covered and the students labelled “control” and “patient” blood with anti-CD3, 4, 8, 14, 19 and 66 mAbs. In Wk 2, students were provided with data files where the levels of major cell populations had been manipulated using magnetic beads to mimic immune diseases states. Using written and video instructions for the software and supplied reference ranges, and using a PC, were asked to identify the disease status of their patient sample. Student perceived understanding of FC was found to significantly increase after the practical session, and student feedback was also overwhelmingly positive. The manipulation of cell populations in whole blood to mimic disease states is an excellent way to teach undergraduate students the major principles of FC. These include labelling of cells, learning to use the software, interpreting the data and disease diagnosis. We will continue to expand our list of disease conditions. All data files are freely available to any teaching staff who may wish to use them as part of their teaching.

Measurement of cytokine release at the single cell level using the ELISPOT assay

The enzyme-linked immunospot (ELISPOT) assay took its design concept from traditional ELISA techniques and evolved over the years from a method for detecting antibodies secreted from B cells to a method for detecting cytokines or other soluble mediators secreted from a variety of different cell types. The ELISPOT assay allows the quantitative measurement of frequency of cytokine secreting cells at the single cell level directly ex vivo without elaborate in vitro expansion or manipulation of cell populations. The function of cells can be inferred from the pattern of cytokines secreted by cells in response to diverse antigenic stimuli and thus the ELISPOT assay has become a powerful method for monitoring immune responses in health and disease. The ELISPOT assay like the ELISA assay is relatively easy to perform and it has the promise of robustness, reliability, and reproducibility of performance for use as a diagnostic tool. The history, applications, validation process, and future challenges of the ELISPOT assay are discussed in this chapter.