Alum-precipitated NP-Ficoll Immunization Protocol

To immunize mice, 4-hydroxy-3-nitrophenol conjugated to AECM-Ficoll (NP-Ficoll, Biosearch Technologies) was alum-precipitated by mixing (1 mg/ml) with an aluminum hydroxide solution at a 1:1 ratio. Precipitation in Alum makes hapten-Ficoll a modestly stronger antigen54 (link). Mice were immunized i.p. with 376 μg per 100 gram body weight. After 7 days, mice were killed and single-cell suspension of the spleen was analyzed by flow cytometry and ELISPOT assay performed as previously described55 (link). For presence anti-NP antibodies, mice we immunized as afore-described and blood collected every 7 days.

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Minguet S., Kläsener K., Schaffer A.M., Fiala G.J., Osteso-Ibánez T., Raute K., Navarro-Lérida I., Hartl F.A., Seidl M., Reth M, & Del Pozo M.A. (2017). Caveolin-1-dependent BCR nanoscale organization regulates B cell tolerance. Nature immunology, 18(10), 1150-1159.

Publication 2017

NP-Ficoll

4 nitrophenol Alum Aluminum hydroxide Anti antibodies Assay Blood Body weight Cell Elispot Ficoll Flow cytometry Hapten Mice Spleen

Corresponding Organization : University of Freiburg

Other organizations : University Medical Center Freiburg

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Variable analysis

independent variables

Immunization of mice with 4-hydroxy-3-nitrophenol conjugated to AECM-Ficoll (NP-Ficoll), alum-precipitated

dependent variables

Immune response of mice, measured by flow cytometry and ELISPOT assay
Presence of anti-NP antibodies in blood, measured every 7 days

control variables

Body weight of mice (376 μg per 100 gram body weight)

controls

Positive control: Not explicitly mentioned
Negative control: Not explicitly mentioned

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