Selectin-mediated lymphocyte homing assay

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Category

Matrices & cellular trafficking

Protocol Name

Authors

Hitomi Hoshino
-, National Center for Geriatrics and Gerontology

Kenji Uchimura *
-, National Center for Geriatrics and Gerontology

*To whom correspondence should be addressed.

KeyWords

Sulfotransferase Lymphocyte Sulfated carbohydrate Homing L-selectin

Reagents

●	PBS (Phosphate Buffered Saline)
●	0.83% NH₄Cl
●	CMFDA (5-chloromethylfluorescein diacetate; Molecular Probes)

Instruments

●	Water bath incubator, 37 °C
●	Flow cytometer (FACSCalibur; Beckton Dickinson)

Methods

Preparation of lymphocytes

1)	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.	Comment 0

2)	Mush the lymph nodes with two glass slides to have a single-cell suspension in pre-warmed PBS.	Comment 0

3)	Filter the suspension with a nylon cell strainer (40 µm pore size; BD Falcon) and collect cells in a 15 mL tube.	Comment 0

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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Labeling of lymphocytes

1)	Add 4.5 µL of 10 mM CMFDA into the suspension. Metabolically label cells by incubating the tube at 37 °C for 30 min.	Comment 0

Harvest the cells as a pellet by centrifugation and then resuspend in pre-warmed PBS at density of 1.7 x 10⁸ cells/ml.

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Intravenous injection

1)	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).	Comment 0

2)	One hour after injection, sacrifice the mice and take out lymphoid organs (peripheral lymph nodes, mesenteric lymph nodes, Peyer’s patches and spleen).	Comment 0

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

1)	Determine the fraction of CMFDA-labeled lymphocytes in suspensions by flow cytometry (5 x 10⁵ cells per organ).	Comment 0

Acquire and analyze data with CellQuest software (Beckton Dickinson).

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Notes

It is important to suspend cell pellets gently when you prepare lymphocytes.
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.
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.
For the L-selectin inhibition experiment, 1 x 10⁶ cells of CMFDA-labeled cells are pre-incubated for 5 min with 1 µg of MEL-14 (anti-Mouse L-selectin; eBioscience) or rat IgG2a (eBioscience). Then, the mixture of cells and antibody are injected intravenously into recipient mice.

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.

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Date of registration：2011-09-16 14:55:31