Intravital two-photon imaging of adoptively transferred B lymphocytes in inguinal lymph nodes

Intravital two-photon imaging allows the observation of immune cells in intact organs of live animals in real time. Recently, several studies using two-photon microscopy have detailed the motility of mouse B and T lymphocyte within lymph nodes and have shown a dependence upon chemokine receptor signaling for the basal velocity of the cells. For, example, T cells from Gnia2 (-/-)mice, deficient in the heterotrimer G-protein G alpha subunit G(alpha i2) have markedly impaired chemokine-triggered chemotaxis. In vivo these cells have reduced motility and impaired positioning within lymph nodes. Gnia2 (-/-) B cells exhibit similar defects. In addition, B cells from Rgs1 (-/-) mice, deficient in a major negative regulator of G(alpha i), have a more robust motility than do wild-type B cells. Here, we describe procedures for visualizing the behavior of fluorescently labeled and adoptively transferred B lymphocytes within the inguinal lymph node of live mice.