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Journal of Immunology Research
Volume 2015, Article ID 129682, 14 pages
Research Article

Infusion of Sulfosuccinimidyl-4-[N-maleimidomethyl]cyclohexane-1-carboxylate-Conjugated MOG35–55-Coupled Spleen Cells Effectively Prevents and Reverses Experimental Autoimmune Encephalomyelitis in Mice

1Department of Hematology, Xuanwu Hospital, Capital Medical University, Beijing 100053, China
2Department of Pathology, Immunology and Laboratory Medicine, University of Florida College of Medicine, Gainesville, FL 32610, USA

Received 27 April 2015; Revised 12 June 2015; Accepted 16 June 2015

Academic Editor: Ethan M. Shevach

Copyright © 2015 Lanfang Zhang et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Supplementary Material

Supplemental Figure 1. The reaction scheme for conjugating MOG and spleen cells with Sulfo-SMCC. In the first reaction, a primary amine on a MOG’s lysine resdues or N-terminus nucleophilically attacks SMCC, release sulfo-NHS, and thus forms a stable amide bond. In the second reaction, a cell surface sulfhydryl reacts with a maleimide functional group on the activated peptide, forming a viable protein-cell conjugate.

Supplemental Figure 2. Thiol (-SH) expression on different subsets of splenocytes and confirmation by 2-Mercaptoethanol neutralization. Freshly prepared splenocytes were incubated for 30 min with Fluorescein-5-maleimide, or Fluorescein-5-maleimide pre-incubated with 2-Mercaptoethanol (2-ME) in 1:2 molar ratio for 30 min. Then, the cells were stained with anti-CD4, CD8, B220, CD11b, CD11c and F4/80 fluorescent antibodies, respectively. The levels of mean fluorescent intensity (MFI) of Fluorescein-5-maleimide represent the levels of thiols on the cell surface. The results showed that the major populations of splenocytes expressed high-level thiols. Pre-incubation of Fluorescein-5-maleimide with 2-ME dramatically attenuated its binding to the cells, suggesting that Fluorescein-5-maleimide was truly reacting with the thiols on the cells surface.

  1. Supplementary Material