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

Published: February 29, 2024

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</…

開示

The authors have nothing to disclose.

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

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記事を引用
A CFSE-Based Assay to Monitor Skin Dendritic Cell Migration to Draining Lymph Nodes in Infected Mice. J. Vis. Exp. (Pending Publication), e21990, doi: (2024).

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