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.
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
2. Mice
3. Immobilization of Animals with Isoflurane Gas Anesthesia
4. BCG Injections and Recovery
5. CFSE Injections
6. Surgical Removal of Popliteal Lymph Node (pLN)
NOTE: Use of sterilized surgical instruments and their repeated decontamination in 70% ethanol are encouraged throughout this step.
7. Generation of Single-cell Suspension from pLN
8. Cell Staining and Flow Cytometry
The authors have nothing to disclose.
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 |