IgE knock-in mice suggest a role for high levels of IgE in basophil-mediated active systemic anaphylaxis
Version of Record online: 27 MAR 2013
© 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
European Journal of Immunology
Volume 43, Issue 5, pages 1231–1242, May 2013
How to Cite
Lübben, W., Turqueti-Neves, A., Okhrimenko, A., Stöberl, C., Schmidt, V., Pfeffer, K., Dehnert, S., Wünsche, S., Storsberg, S., Paul, S., Bauer, S., Riethmüller, G., Voehringer, D. and Yu, P. (2013), IgE knock-in mice suggest a role for high levels of IgE in basophil-mediated active systemic anaphylaxis. Eur. J. Immunol., 43: 1231–1242. doi: 10.1002/eji.201242675
- Issue online: 25 APR 2013
- Version of Record online: 27 MAR 2013
- Accepted manuscript online: 19 FEB 2013 12:04PM EST
- Manuscript Accepted: 14 FEB 2013
- Manuscript Revised: 16 JAN 2013
- Manuscript Received: 11 MAY 2012
- DFG. Grant Number: Yu 47/1-1
- ERC. Grant Number: PAS_241506
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Figure S1 Gating strategies for FACS analysis and summary of surface IgG1+ and IgE+ stainings. Upper panels gating strategy for Nb infection. Right upper graph shows % IgG1+ cells of total lymphocytes, the lower right graph shows % IgE+ cells of total lymphocytes, statistical analysis was done using the student´s t test. Middle panels show in vitro stimulated spleen cells (LPS or LPS+IL-4). Upper, middle right panel shows percent IgE + cells and lower panel shows percent IgG1 positive cells (see also Fig. 2A) Lower panels show gating strategy for CD23 and IgE expression of CD45RB/B220 positive spleen cells (Fig. 2C).
Figure S2 Depletion of basophils in wild type and IgE knock in mice. Exemplary FACS of peripheral blood cells of naïve mice treated with 30μg Ba103 (in 100μl PBS) or PBS alone i.p. injection 24h before. Over 90% of basophils (CD49b+, IgE+) are depleted . Right panels TNP-OVA immunized and boosted mice were injected with 30μg Ba103 (in 100μl PBS) or PBS alone 24h before FACS analysis. In wild type, heterozygous and homozygous IgEki mice 65%, 80% and 85% of basophils (CD49b+-IgE+) in peripheral blood were depleted, respectively. n=2, single experiments.
Figure S3 Sequence comparison between IgE knock in targeting vector (Construct), published Balb/c and C57BL/6 sequences of the IgE heavy chain. The difference between Construct and Balb/c are marked yellow and between Balb/c/Construct and C57BL/6 are marked in blue. The analysis suggests that the IgE knock in is of allotype IgEa, derived from a genomic clone of 129Sv. Balb/c is also IgEa.
Figure S4 As a PCR control we cloned a similar genomic fragment of the IgG1 region in front of IgE. The test-arm fragment was 155bp longer, as the actual target vector region, in order to avoid PCR contaminations. The expected PCR size for controls is 1050bp and for correct integration of the target vector is 895bp. Left Sequence depicts the control PCR template (Test arm) and right sequence the PCR part of the Gene Targeting vector (short arm). Cloning vector sequences (red), IgG1 sequences (black), screening PCR primers (cyan), IgE sequences (green), the 5-prime end of the Gene Targeting vector (magenta) and the additional IgG1 sequences in the control PCR construct (blue) are marked.
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