Selectin-mediated lymphocyte homing assay
Lymphocytes circulate between the blood and secondary lymphoid organs. Lymphocyte homing to lymph nodes and Peyer’s patches is initiated by the binding of L-selectin on lymphocytes to its ligands expressed in high endothelial venules (HEVs). A major class of L-selectin ligands in HEVs is sialyl 6-sulfo Lewis X. GlcNAc-6-sulfation catalyzed by GlcNAc-6-sulfotransferases is essential for the ligand activity. In this section, we describe an in vivo assay for quantifying lymphocyte homing to lymph nodes in mice.
PBS (Phosphate Buffered Saline)
CMFDA (5-chloromethylfluorescein diacetate; Molecular Probes)
Water bath incubator, 37 °C
Flow cytometer (FACSCalibur; Beckton Dickinson)
Preparation of lymphocytes
Dissect out mesenteric lymph nodes of 5- to 16-week-old C57BL/6 mice and place them in a plastic plate containing pre-warmed PBS.
Mush the lymph nodes with two glass slides to have a single-cell suspension in pre-warmed PBS.
Filter the suspension with a nylon cell strainer (40 µm pore size; BD Falcon) and collect cells in a 15 mL tube.
Harvest the cells as a pellet by centrifugation at room temperature and then resuspend in 9 mL of pre-warmed PBS.
Labeling of lymphocytes
Add 4.5 µL of 10 mM CMFDA into the suspension. Metabolically label cells by incubating the tube at 37 °C for 30 min.
Harvest the cells as a pellet by centrifugation and then resuspend in pre-warmed PBS at density of 1.7 x 108 cells/ml.
1.7 x 107 cells of CMFDA-labeled lymphocytes in 100 µl of PBS are injected into the tail vein of a recipient mouse by a 1 mL syringe with a 29-gauge needle (Beckton Dickinson).
One hour after injection, sacrifice the mice and take out lymphoid organs (peripheral lymph nodes, mesenteric lymph nodes, Peyer’s patches and spleen).
Place the organs in cold PBS. Create single-cell suspensions of these organs in PBS by teasing them with a 23-gauge needle.
Quantification of labeled cells by flow cytometry
Determine the fraction of CMFDA-labeled lymphocytes in suspensions by flow cytometry (5 x 105 cells per organ).
Acquire and analyze data with CellQuest software (Beckton Dickinson).
Figure & Legends
Figure 1 L-selectin-mediated lymphocyte homing to lymphoid organs of wild-type
and GlcNAc6ST-1/GlcNAc6ST-2 double-knockout mice.
Homing of CMFDA-labeled mesenteric lymphocytes was quantified. Cells from mice were preincubated with MEL-14 or isotype-matched control (IgG2a) for 5 min before transfer into recipient mice (n=3 each genotype). The percentage of labeled cells accumulating in peripheral lymph nodes (PLN), mesenteric lymph nodes (MLN), Peyer’s patches (PP) and spleens (SPL) was determined in wild-type (WT) and GlcNAc6ST-1/GlcNAc6ST-2 double-knockout mice.
This figure was originally published in Nat Immunol. Uchimura K. et al. “A major class of L-selectin ligands is eliminated in mice deficient in two sulfotransferases expressed in high endothelial venules” 2005, 6(11):1105-13, doi:10.1038/ni1258.
Hemmerich S, Bistrup A, Singer MS, van Zante A, Lee JK, Tsay D, Peters M, Carminati JL, Brennan TJ, Carver-Moore K, Leviten M, Fuentes ME, Ruddle NH, Rosen SD (2001) Sulfation of L-selectin ligands by an HEV-restricted sulfotransferase regulates lymphocyte homing to lymph nodes. Immunity 15: 237-247. [PMID : 11520459]
Uchimura K, Kadomatsu K, El-Fasakhany FM, Singer MS, Izawa M, Kannagi R, Takeda N, Rosen SD, Muramatsu T. (2004) N-acetylglucosamine 6-O-sulfotransferase-1 regulates expression of L-selectin ligands and lymphocyte homing. J Biol Chem. 279:35001-35008. [PMID : 15175329]
Uchimura K, Gauguet JM, Singer MS, Tsay D, Kannagi R, Muramatsu T, von Andrian UH, Rosen SD (2005) A major class of L-selectin ligands is eliminated in mice deficient in two sulfotransferases expressed in high endothelial venules. Nat Immunol. 6: 1105-1113. [PMID : 16227986]