Biotinylation Assay to Determine LFA-1 Recycling in Motile T-Lymphocytes
The cycles of internalization of the cell surface β2 integrin receptor lymphocyte function-associated antigen 1 (LFA-1) and its re-exposure on the plasma membrane are important for T-cell trafficking. Biotinylation of cells enables to measure surface expression of receptors, and after reducing surface biotin with reducing buffer, enables to measure the internalization of receptors. Here, by using biotin in combination with reducing buffer and recombinant intercellular adhesion molecule-1 (rICAM-1)-coated dishes and subsequent Western immunoblot analysis, we describe how to measure internalization of the LFA-1 receptor and its re-expression back to the cell surface in motile T-lymphocytes.
Key wordsLFA-1 recycling Biotinylation T-cell migration
LFA-1m38 was a kind gift from Dr. Nancy Hogg (The Francis Crick Institute, London, UK). This work was supported by Swedish Research Council awards K2010-80P-21592-01-4 and K2010-80X-215917-01-4, Foundation Olle Engquist Byggmästare, I&A Lundberg Research Foundation, Royal Swedish Academy of science, Royal Physiographic Society of Lund, Åke Wiberg, Jeanssons Foundation, Kocks Foundation, P&U Schybergs Foundation, Gyllenstiernska Krapperup Foundation, Gustav V 80 Jubilee Fund, Österlund Foundation, Nanna Svartz and Crafoord awards.