Tool	Description	Selected reference
Cannulation studies	This procedure involves the surgical insertion of cannula (tube) directly into an afferent or efferent vessel or into the cisterna chyli, to collect lymph fluid. The cellular composition of lymph is subsequently analyzed, typically by flow cytometry or microscopy methods	(5–9)
Adoptive transfer	In adoptive transfer experiments, cells are isolated from donor mice, fluorescently labeled (unless already marked by endogenous expression of a fluorophore or a congenic marker) and intravenously or subcutaneously injected into a recipient mouse. In some cases, T cells are subjected to an in vitro culturing step (e.g., in vitro activation) prior to injection. At defined time points after transfer, T cell numbers in lymph nodes (LNs) (or other tissue) are quantified by flow cytometry, LN sectioning and microscopy, or other means. While this experimental setup is technically straightforward, the transferred cells may differ from the endogenously migrating populations. Also, typically only a small fraction of cells injected subcutaneously actually migrate to dLNs or beyond	(7, 10–13)
Intravital microscopy (IVM)	This technique allows the study of migratory processes at the single-cell level and in real time. It involves fluorescence-based time-lapse imaging by, e.g., confocal-/multiphoton- or stereomicroscopy. Several mouse reporter lines expressing a fluorescent protein in lymphatic vessels (LVs) have been generated (14–18). In the case of T cells, most studies have been performed with fluorescently labeled and adoptively transferred T cells, but endogenous models are also available (19–21)	(22–26)
Intralymphatic injection	Microinjection of T cells directly into a LV upstream of a draining lymph node. Similar to adoptive transfer but permits the study of T cell entry specifically across the LN subcapsular sinus. This represents an elegant yet technically challenging method complementing IVM studies	(25)
LN egress studies	This experimental setup allows quantifying dwell time of T cells in LNs. In a typical experiment, fluorescently labeled T cells are first transferred intravenously into a recipient mouse. After an equilibration phase, further T cell ingress into LNs is blocked by administration of entry-blocking antibodies (e.g., directed against the integrin subunit α4 or against L-selectin). Antibody treatment allows the uncoupling of T cell entry from exit, which continues to occur. Exit rates, for example, can be calculated by comparing fluorescent T cell numbers in LNs at the time of antibody injection to a later time point (e.g., 24 h later; flow cytometry-based quantification)	(9, 22, 27, 26)
Photoconvertible transgenic mice	The use of photoconvertible transgenic mice permits monitoring the migration of endogenously labeled cells in vivo. It requires transgenic mice expressing a photoconvertible fluorescent protein in all cell types [e.g., Kaede protein (28) or Kikume Green–Red protein (29)]. Upon illumination with violet light, fluorescent proteins undergo irreversible changes that alter their fluorescent spectrum (typically a green to red shift). By selectively illuminating the tissue at a particular site (e.g., skin), one can subsequently quantify the appearance of photoconverted T cells in other tissues (e.g., dLNs) to gain insight about their trafficking behavior. The system can easily be combined with pharmacologic blockade of genes of interest. Alternatively, backcrossing onto a genetic knockout can be done	(28, 30, 31)