A CFSE-Based Assay to Monitor Skin Dendritic Cell Migration to Draining Lymph Nodes in Infected Mice

Abstract

Source: Bollampalli, V. P., et al. A CFSE-based Assay to Study the Migration of Murine Skin Dendritic Cells into Draining Lymph Nodes During Infection with Mycobacterium bovis Bacille Calmette-Guérin. J. Vis. Exp. (2016)

This video demonstrates an assay for monitoring dendritic cell migration following footpad infection with Mycobacterium bovis Bacille Calmette-Guérin BCG in mice. Upon bacterial infection, activated skin DC cells are labeled with a cell-permeable carboxyfluorescein diacetate succinimidyl ester (CFSE) dye. After a suitable interval for DC migration to the popliteal draining lymph node, the node cells are isolated and subjected to immunostaining for DC-specific surface markers. Identification of migrated DCs is performed using flow cytometry.

Protocol

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board. 1. Preparation of CFSE Stocks and Storage Prepare a 5 mM stock of 5- and 6-carboxyfluorescein diacetate succinimidyl ester (CFSE) in dimethyl sulfoxide. Vortex. Dispense 100 µl aliquots into sterile, 0.5 ml screw cap microtubes. Store aliquots at -80 °C. 2. Mice</…

Materials

Isoflurane	Baxter	1001936060
5- and 6-carboxyfluorescein diacetate succinimidyl ester (CFSE)	Invitrogen	C1157
Dimethyl sulphoxide (DMSO)	Sigma	D2650
Fetal bovine serum (FBS)	Sigma	F7524
Bacille Calmette-Guérin (BCG)			Strain Pasteur, in house
Trypan blue solution	Sigma	T8154
EDTA			In house
Sodium azide	Sigma	S2002
PBS			In house
Filtered water			In house
BD Plastipak 3 ml syringe	BD Medical Surgical Systems	309658	Alternative: 5 ml syringe
6 well plates	TPP	92006
15 ml centrifuge tubes	TPP	91015	Polypropylene
5 ml round bottom tubes	BD Falcon	3272948	Polystyrene
Univentor 400 anaesthesia unit	Univentor	8323001
Monojet 3/10ml syringe	Medicarrier	8881511144	29 G x 1/2 in.
Sterile filter unit	TPP	99500	PES membrane
EPPI-Pistill homogenizer	Schuett biotech	3.200 512	Polypropylene
Allegra X-30R centrifuge	Beckman Coulter	B01145
Bürker counting chamber	VWR	631-0920
0.5 ml screw cap microtube	Sarstedt	72.73	Polypropylene
1.5 ml Eppendorf microcentrifuge tube	VWR	700-5239	Polypropylene
70 micron cell strainers	VWR	734-0003
FlowJo cell analysis software	Tree Star
Antibody	Clone	Company
Antibodies
MHC-II I-A/I-E	M5/114.15.2	BD Biosciences
CD11b	M1/70	BD Biosciences
CD11c	HL3	BD Biosciences
CD16/CD32 (Fc-block)	2.4G2	BD Biosciences
CD326/EpCAM	G8.8	Biolegend
CD103	2.00E+07	Biolegend
CD80	16-10A1	Biolegend
CD86	GL-1	Biolegend