Abstract
Determining the identity of cells of the immune system usually involves destructive fixation and chemical staining, or labeling with fluorescently labeled antibodies recognising specific cell surface markers. Completely label-free identification would be a significant advantage in conditions where untouched cells are a priority. We demonstrate here the use of Wavelength Modulated Raman Spectroscopy, to achieve label-free identification of purified, unfixed and untouched populations of major immune cell subsets isolated from healthy human donors. Using this technique we have been able to distinguish between CD4+ T lymphocytes, CD8+ T lymphocytes and CD56+ Natural Killer cells at specificities of up to 96%. Additionally, we have been able to distinguish between CD303+ plasmacytoid and CD1c+ myeloid dendritic cell subsets, the key initiator and regulatory cells of many immune responses. This demonstrates the ability to identify unperturbed cells of the immune system, and opens novel opportunities to analyse immunological systems and to develop fully label-free diagnostic technologies.
Highlights
The mammalian immune system comprises distinct bone marrow-derived cell types that interact to provide protection against an extensive array of potential pathogens including bacteria, viruses, fungi and parasites
To address all three of these aspects, we demonstrate the use of Wavelength Modulated Raman Spectroscopy (WMRS) for the first time on a tunable Ti:Sapphire laser to distinguish between CD4+, CD8+ T cells and CD56+ Natural Killer (NK) cells
CD4+ T lymphocytes were obtained at a purity level typically up to 96.5%, and secreted high levels of the cytokine IL-2 in response to incubation with beads coupled with anti-CD3 and— CD28 antibodies (Fig 1A and 1B)
Summary
The mammalian immune system comprises distinct bone marrow-derived cell types that interact to provide protection against an extensive array of potential pathogens including bacteria, viruses, fungi and parasites. Monitoring changes in the numbers of these cells in human blood can indicate the presence of inflammation and infection. In humans the population of lymphocytes known as T cells can be divided into two main groups based upon their expression of CD4 and CD8 cell surface proteins[1]. CD4+ T cells usually function through the secretion of bioactive cytokines [2], whereas CD8+ T cells are typically known as cytotoxic T cells, which can directly kill virally infected cells [3]. A PLOS ONE | DOI:10.1371/journal.pone.0125158 May 20, 2015
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