Christian Möbs, Thomas Schmidt Journal of Investigative Dermatology

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ELISPOT Assay: Cytokine

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Research Techniques Made Simple: Monitoring of T-Cell Subsets using the ELISPOT Assay Christian Möbs, Thomas Schmidt Journal of Investigative Dermatology Volume 136, Issue 6, Pages e55-e59 (June 2016) DOI: 10.1016/j.jid.2016.04.009 Copyright © 2016 The Authors Terms and Conditions

Figure 1 ELISPOT principle. (a) Coating of ELISPOT membranes (microwells) with primary (capture) antibody. (b) Blocking of unspecific binding sites by saturation of the membrane with protein solution (e.g., phosphate buffered saline + 10% fetal calf serum). (c) Addition of a single-cell suspension and activation of cells to secrete analytes (red circles) using an appropriate stimulus. (d) Analyte binding to the specific capture antibody followed by removal of cells. (e) Addition of a secondary biotinylated (detection) antibody, which binds to the respective analyte-antibody complex. (f) Binding of avidin- or streptavidin-conjugated enzyme (e.g., horseradish peroxidase) to the biotinylated detection antibody. (g) Addition of chromogenic substrate solution and visualization of spots by enzymatic substrate transformation. (h) Representative image of an ELISPOT membrane that can be automatically analyzed by an ELISPOT reader. Journal of Investigative Dermatology 2016 136, e55-e59DOI: (10.1016/j.jid.2016.04.009) Copyright © 2016 The Authors Terms and Conditions

Figure 2 IFN-γ ELISPOT assay from IL-2–expanded TILs after peptide stimulation. Shown are background counts of unstimulated TILs (–) and TILs incubated with “irrelevant peptides,” (1) Bcl-X(L) 165–173 (RIAAWMATY), (2) Rho1 (RAGLQVRKNK), and (3) Rho1L2 (RLGLQVRKNK), which did not induce IFN-γ secretion compared with the specific IFN-γ response after stimulation with the survivin-derived epitope SUR53-62 (DLAQCFFCFK). The bars of spot-forming cells represent the number of unstimulated (–) and peptide-stimulated TILs. Respective raw data are depicted by images of ELISPOT membranes in duplicate (reprinted from Junker et al., 2012, with permission from Elsevier). SFC, spot-forming cell; TIL, tumor-infiltrating lymphocyte. Journal of Investigative Dermatology 2016 136, e55-e59DOI: (10.1016/j.jid.2016.04.009) Copyright © 2016 The Authors Terms and Conditions

Figure 3 Stimulus-dependent cytokine secretion of PBMCs from patients with AD and healthy control subjects. (a, b) IFN-γ and (c, d) IL-4 ELISPOT assay of ex vivo PBMCs activated with either (a, c) PHA or (b, d) SEB in patients with AD (black lines) and in control subjects (gray lines). (e) Ex vivo PBMC IL-4 ELISPOT stimulated with house dust mite extract. AD, n = 18; control, n = 10. ∗P < 0.05. (reprinted from Newell et al., 2013, with permission from Elsevier). AD, atopic dermatitis; PBMC, peripheral blood mononuclear cell; PHA, phytohemagglutinin; SEB, staphylococcal enterotoxin B; SFU, spot-forming units. Journal of Investigative Dermatology 2016 136, e55-e59DOI: (10.1016/j.jid.2016.04.009) Copyright © 2016 The Authors Terms and Conditions