Table 1.

Methods for studying leukocyte migration

Method	Description, advantages (+) and disadvantages (−)
Cannulation studies Used to study: Lymph node (LN) exit (efferent lymph) Entry into afferent lymphatic vessels (LVs) (afferent lymph)	Lymph fluid is collected via a cannula placed directly inside an afferent or efferent LV, or into the cisterna chyli. Its cellular content is subsequently characterized by microscopy or flow cytometry. Cannulation studies are typically performed in larger animals/humans, and more rarely also in rodents (Gowans 1957; Mackay et al. 1988; Yawalkar et al. 2000; Zawieja et al. 2019). + Allows to characterize endogenous migration into afferent/efferent LVs. + Multiple types of analyses possible, ranging from cellular characterization to different -omics techniques. − Technically challenging, especially when performed in small animals (mice).
Adoptive transfer (I) (e.g., injected subcutaneously or into the footpad) Used to study: Entry into afferent LVs Entry from afferent LVs into LNs	Fluorescently labeled or otherwise marked leukocytes (e.g., expressing a fluorescent protein or congenic marker) are injected into peripheral tissues of recipient mice and their presence in the draining LN (dLN) is subsequently quantified by flow cytometry or immunofluorescence. Common sites of injection are the ear skin or footpad, as they mainly drain into one single LN (i.e., the auricular or popliteal LN, respectively). In some cases, intralymphatic injection is also used (Ohl et al. 2004; Johnson et al. 2006; Brinkman et al. 2016; Teijeira et al. 2017; Martens et al. 2020). To study the residence time of T cells in LNs, a special adoptive transfer setup is used. Specifically, fluorescently labeled T cells are first transferred intravenously into a recipient mouse from where they home to LNs. After a few hours, further immigration of T cells into LNs via high endothelial venules (HEVs) is blocked by administration of entry-blocking antibodies (e.g., anti-integrin α4 or anti-L-selectin). In this way, T-cell entry is uncoupled from egress, which will continue to occur. To quantify LN egress, the number of fluorescent T cells in the LNs at the time of antibody injection is determined by quantitative flow cytometry and compared to the number found in the LNs in the control and the treatment group (or wild-type [WT] vs. knockout [KO]) at a certain end point (e.g., 24 h). If egress is inhibited, more T cells should be present in the LN in the treated (or KO) group at the end point (Halin et al. 2005; Pham et al. 2010; Reichardt et al. 2013). + Useful to study the role of a gene of interest in leukocyte migration in vivo. − Adoptively transferred cells need to be injected in high numbers. Often cells are generated by in vitro expansion or activation, which might not always reflect the phenotype of endogenous leukocytes. − When assessing migration through afferent LVs: Cell injection can damage the tissue at the injection site and push the leukocytes into LVs.
Adoptive transfer (II) Intravenous injection Used to study LN egress
FITC/TRITC painting Used to study: Entry into afferent LVs Entry from afferent LVs into LNs	Topical or intranasal/intratracheal administration of fluorescein isothiocyanate (FITC) or TRITC allows analysis of the endogenous migration of DCs from the skin or lung, respectively. FITC or TRITC is taken up by dermal or pulmonary DCs, which subsequently can be identified in dLNs by flow cytometry or microscopy (Förster et al. 1999; Qu et al. 2004; Vigl et al. 2011; Iolyeva et al. 2013). + Allows the study of endogenous DC migration via afferent LVs. − Method only works for phagocytic cells like DCs, but not for lymphocytes. − FITC and TRITC act as contact sensitizers, and application may lead to light irritation of the tissue that might be suboptimal when studying migration in steady-state.
Photoconvertible transgenic mice Used to study: Migration via afferent LVs to LNs	Performed in mice ubiquitously expressing a photoconvertible green- fluorescent protein (e.g., Kikume or Kaede protein), which is converted to a red-fluorescent state upon illumination with (ultra)violet light. Migratory photoconverted cells can be tracked by their red fluorescence and detected and quantified by flow cytometry in tissues like LNs (Tomura et al. 2008, 2010; Bromley et al. 2013; Tadayon et al. 2021). + Allows one to investigate migration of endogenous leukocytes and to identify different subsets of migrating cells. − Migration from internal organs (e.g., LNs) can only be studied after invasive surgery (required for illumination).
Intravital microscopy Used to study: Entry/migration within afferent LVs Entry into or exit from LNs across lymphatic sinuses	Fluorescent reporter mice adoptively transferred fluorescently labeled leukocytes or injected fluorescent antibodies are used to visualize LVs and leukocytes in vivo. The migration of leukocytes is studied at the single-cell level by time-lapse confocal or multiphoton microscopy to determine (e.g., the speed, directionality, or cellular interactions of migrating cells). Common imaging sites are the popliteal LNs, ear skin, or footpads (Grigorova et al. 2010; Tal et al. 2011; Teijeira et al. 2017; Hunter et al. 2019). + Allows the identification and/or exploration of distinct steps in the overall migration process. + Delivers complementary information to methods investigating cell migration at the population level (e.g., photoconversion or adoptive transfer studies). − Not every tissue can be imaged with minimal invasiveness (e.g., LNs need to be surgically exposed in anesthetized mice and this can induce tissue inflammation). − The creation of double- or triple-transgenic mice with multiple fluorescent cellular reporters is time-consuming.
Skin explants Used to study: Migration toward, into, and within afferent LVs	Murine ear skin is ripped along the central cartilage and fluorescently labeled leukocytes are added on top of the luminal side of the explant and left to migrate into the dermal tissue. Migration into afferent lymphatics is analyzed after ∼1–4 h by confocal microscopy after staining LVs with fluorescent antibodies. Alternatively, migration of fluorescent leukocytes can be assessed in real time by performing time-lapse imaging in explants with fluorescent LVs (either stained by antibodies or endogenously visualized in LV reporter mice) (Lämmermann et al. 2008; Teijeira et al. 2013; Russo et al. 2016; Johnson et al. 2017). + Intermediate between in vitro and in vivo. Allows one to investigate a distinct migration step (e.g., chemotaxis, transmigration or intralymphatic crawling) in a complex tissue environment. − The ripping of the ear induces a proinflammatory response, up-regulating many inflammatory genes (e.g., adhesion molecules in LVs). − Soluble mediators (e.g., chemokines) can diffuse out of the ripped tissue, and lymph flow is absent.
In vitro assays Used to study: Adhesion Transmigration Crawling	Specific migration steps, such as transmigration, adhesion, or crawling, can be investigated by in vitro functional assays using lymphatic endothelial cell (LEC) monolayers and ex vivo isolated or in vitro differentiated/activated leukocytes (Ledgerwood et al. 2008; Russo et al. 2016; Arasa et al. 2021; Johnson et al. 2021). + Very useful for investigating the involvement of a potential target gene in lymphatic migration before moving to more expensive and technically challenging in vivo experiments. − Simplified assay system in which the composition of lymphatic capillaries (e.g., junctional arrangement) is not maintained. − Expression of gene of interest may be lost in in vitro cultured cells.