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. |
Category | Matrices & cellular trafficking |
Protocol Name | Selectin-mediated lymphocyte homing assay |
Authors
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Hoshino, Hitomi
University of Fukui, Faculty of Medical Sciences
Uchimura, Kenji
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Nagoya University Graduate School of Medicine, Department of Biochemistry
*To whom correspondence should be addressed.
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KeyWords |
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Reagents
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PBS (Phosphate Buffered Saline) |
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CMFDA (5-chloromethylfluorescein diacetate; Thermo Fisher Scientific Inc., Waltham, MA) |
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Instruments
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Water bath incubator, 37°C |
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Flow cytometer (FACSCalibur: BD, Franklin Lakes, NJ) |
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Methods |
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Preparation of lymphocytes
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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. |
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Mush the lymph nodes with two glass slides to have a single-cell suspension in pre-warmed PBS. |
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Filter the suspension with a nylon cell strainer (40 μm pore size: BD) and collect cells in a 15 mL tube. |
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Harvest the cells as a pellet by centrifugation at room temperature and then resuspend in 9 mL of pre-warmed PBS. |
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Add 4.5 μL of 10 mM CMFDA into the suspension. Metabolically label cells by incubating the tube at 37°C for 30 min. |
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Harvest the cells as a pellet by centrifugation and then resuspend in pre-warmed PBS at density of 1.7 × 108 cells/mL. |
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1.7 × 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 (BD). |
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One hour after injection, sacrifice the mice and take out lymphoid organs (peripheral lymph nodes, mesenteric lymph nodes, Peyer’s patches and spleen). |
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Place the organs in cold PBS. Create single-cell suspensions of these organs in PBS by teasing them with a 23-gauge needle. |
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Quantification of labeled cells by flow cytometry
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Determine the fraction of CMFDA-labeled lymphocytes in suspensions by flow cytometry (5 × 105 cells per organ). |
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Acquire and analyze data with CellQuest software (BD). |
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Notes |
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It is important to suspend cell pellets gently when you prepare lymphocytes.
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During the step 1 and 2 pay attention no to change the temperature of cell suspension. Shedding of L-selectin can be triggered by temperature change.
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In the case of preparation of lymphocytes from the spleen, red blood cell lysis should be carried out by suspending the pellet in the step 1-(4) in 0.83% NH4Cl for 2-5 min.
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For the L-selectin inhibition experiment, 1 × 106 cells of CMFDA-labeled cells are pre-incubated for 5 min with 1 μg of MEL-14 (anti-Mouse L-selectin: eBioscience, Inc., San Diego, CA) or rat IgG2a (eBioscience, Inc.). Then, the mixture of cells and antibody are injected intravenously into recipient mice.
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Figure & Legends |
Figure & Legends
Fig. 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. |
Copyrights |
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This work is released underCreative Commons licenses
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Date of registration:2015-10-01 13:38:51 |
- Hemmerich, S., Bistrup, A., Singer, M.S., van Zante, A., Lee, J.K., Tsay, D., Peters, M., Carminati, J.L., Brennan, T.J., Carver-Moore, K., Leviten, M., Fuentes, M.E., Ruddle, N.H., and Rosen, S.D. (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, F.M., Singer, M.S., Izawa, M., Kannagi, R., Takeda, N., Rosen, S.D., and 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, J.M., Singer, M.S., Tsay, D., Kannagi, R., Muramatsu, T., von Andrian, U.H., and Rosen, S.D. (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]
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