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Immunology and Infection

Generering af lymfeknuder-fedt Pad kimærer for Studiet af lymfeknuder stromacelle Origin

Published: December 16, 2013 doi: 10.3791/50952

Materials

Name Company Catalog Number Comments
2-Mercaptoethanol Sigma M3148
50 mm Sterilin Petri Dish Appleton Woods SC265
90 mm Sterilin Petri Dish Appleton Woods SC260
Adhesive slides Surgipath 00202
Anti-mouse CD31 eFluor 450 eBioscience 48-0311
Anti-mouse CD4 Alexa 647 eBioscience 51-0041
Anti-mouse CD45 PerCP-Cy5.5 eBioscience 45-0451
Anti-mouse IgM Rhodamine Red Stratech 715-296-020-JIR
Anti-mouse Podoplanin PE/Cy7 Biolegend 127411
Anti-mouse Podoplanin purified eBioscience 14-5381
Collagenase D Roche 11088858001
DMEM Medium Sigma D5671
DNase I Sigma DN25-1G
Dumont #5 Forceps Inox Biologie FST 11252-20 Extra fine forceps for embryonic and newborn dissections
EDTA solution 0.5 M Sigma E7889
ERTR-7 Biogenesis
Fetal bovine serum Sigma F9665
Hardened fine iris scissors straight 11 cm FST 14090-11 Small scissors for dissection
HEPES solution Sigma H0887
Isopore Membrane Filters 0.8 μm ATTP Millipore ATTPO 1300
L-Glutamine solution Sigma G7513
MEM Nonessential Amino Acid Solution (100x) Sigma M7145
O.C.T. Compound Tissue-Tek 4583
Penicillin-Streptomycin Sigma P4458
Plastic box with lid Watkins and Doncaster E6052 Sandwich box for in vitro organ culture. Drill two holes in the lid to allow gas exchange in the CO2 incubator.
RPMI 1640 Medium Sigma R0883
Sponges Vulkan Underwrap Patterson Medical 004383
Stereomicroscope Leica LEICA MZ95 Dissecting microscope with zoom
Thermomixer Eppendorf 5436
Vectashield Mounting Medium with DAPI Vector Laboratories H-1200

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References

  1. Mueller, S. N., Germain, R. N. Stromal cell contributions to the homeostasis and functionality of the immune system. Nat. Rev. Immunol. 9, 618-629 (2009).
  2. Roozendaal, R., Mebius, R. E. Stromal cell-immune cell interactions. Annu. Rev. Immunol. 29, 23-43 (2011).
  3. Turley, S. J., Fletcher, A. L., Elpek, K. G. The stromal and haematopoietic antigen-presenting cells that reside in secondary lymphoid organs. Nat. Rev. Immunol. 10, 813-825 (2010).
  4. Katakai, T., et al. Organizer-like reticular stromal cell layer common to adult secondary lymphoid organs. J. Immunol. 181, 6189-6200 (2008).
  5. Link, A., et al. Fibroblastic reticular cells in lymph nodes regulate the homeostasis of naive T cells. Nat. Immunol. 8, 1255-1265 (2007).
  6. Benezech, C., et al. Ontogeny of stromal organizer cells during lymph node development. J. Immunol. 184, 4521-4530 (2010).
  7. Cupedo, T., et al. Presumptive lymph node organizers are differentially represented in developing mesenteric and peripheral nodes. J. Immunol. 173, 2968-2975 (2004).
  8. Avan de Pavert, S., Mebius, R. E. New insights into the development of lymphoid tissues. Nat. Rev. Immunol. 10, 664-674 (2010).
  9. Vondenhoff, M. F., et al. LTbetaR signaling induces cytokine expression and up-regulates lymphangiogenic factors in lymph node anlagen. J. Immunol. 182, 5439-5445 (2009).
  10. Dougall, W. C., et al. RANK is essential for osteoclast and lymph node development. Genes Dev. 13, 2412-2424 (1999).
  11. Kim, D., et al. Regulation of peripheral lymph node genesis by the tumor necrosis factor family member TRANCE. J. Exp. Med. 192, 1467-1478 (2000).
  12. Carragher, D., et al. A stroma-derived defect in NF-kappaB2-/- mice causes impaired lymph node development and lymphocyte recruitment. J. Immunol. 173, 2271-2279 (2004).
  13. White, A., et al. Lymphotoxin a-dependent and -independent signals regulate stromal organizer cell homeostasis during lymph node organogenesis. Blood. 110, 1950-1959 (2007).
  14. Benezech, C., et al. Lymphotoxin-beta receptor signaling through NF-kappaB2-RelB pathway reprograms adipocyte precursors as lymph node stromal cells. Immunity. 37, 721-734 (2012).
  15. Anderson, G., Jenkinson, E. J., Moore, N. C., Owen, J. J. MHC class II-positive epithelium and mesenchyme cells are both required for T-cell development in the thymus. Nature. 362, 70-73 (1993).
  16. Szot, G. L., Koudria, P., Bluestone, J. A. Transplantation of pancreatic islets into the kidney capsule of diabetic mice. J. Vis. Exp. (9), e404 (2007).
  17. Krautler, N. J., et al. Follicular dendritic cells emerge from ubiquitous perivascular precursors. Cell. 150, 194-206 (2012).
Generering af lymfeknuder-fedt Pad kimærer for Studiet af lymfeknuder stromacelle Origin
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Cite this Article

Benezech, C., Caamano, J. H.More

Benezech, C., Caamano, J. H. Generation of Lymph Node-fat Pad Chimeras for the Study of Lymph Node Stromal Cell Origin. J. Vis. Exp. (82), e50952, doi:10.3791/50952 (2013).

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