Bi-ligand surfaces with oriented and patterned protein for real-time tracking of cell migration

• A bioactive platform for detailed observation of 2-D cell migration is presented.
• Migration factors are oriented on surface via His- and Fc-tags and IMAC chemistry.
• ICAM-1 surface immobilization along with activation enabled B cell migration.
• No influence of surface immobilized SDF-1α on activated B cell migration was seen.
• Controlled exposure to UV enabled patterning via linear protein gradient fabrication.

A bioactive platform for the quantitative observation of cell migration is presented by (1) presenting migration factors in a well-defined manner on 2-D substrates, and (2) enabling continuous cell tracking. Well-defined substrate presentation is achieved by correctly orienting immobilized proteins (chemokines and cell adhesion molecules), such that the active site is accessible to cell surface receptors. A thiol-terminated self-assembled monolayer on a silica slide was used as a base substrate for subsequent chemistry. The thiol-terminated surface was converted to an immobilized metal ion surface using a maleimido-nitrilotriacetic acid (NTA) cross-linker that bound Histidine-tagged recombinant proteins on the surface with uniform distribution and specific orientation. This platform was used to study the influence of surface-immobilized chemokine SDF-1α and cell adhesion molecule ICAM-1 on murine splenic B lymphocyte migration. While soluble SDF-1α induced trans-migration in a Boyden Chamber type chemotaxis assay, immobilized SDF-1α alone did not elicit significant surface-migration on our test-platform surface. Surface-immobilized cell adhesion protein, ICAM-1, in conjunction with activation enabled migration of this cell type on our surface. Controlled exposure to UV light was used to produce stable linear gradients of His-tagged recombinant SDF-1α co-immobilized with ICAM-1 following our surface chemistry approach. XPS and antibody staining showed defined gradients of outwardly oriented SDF-1α active sites. This test platform can be especially valuable for investigators interested in studying the influence of surface-immobilized factors on cell behavior and may also be used as a cell migration enabling platform for testing the effects of various diffusible agents.

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