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

Overview

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

1. 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

1. Use inbred male or female mice between 8 and 12 weeks of age. Sex- and age-matched animals are preferred. Make sure the necessary animal ethical permits are in place before the initiation of experiments.

3. Immobilization of Animals with Isoflurane Gas Anesthesia

1. Load the anesthesia unit with isoflurane. Fill the 10 ml gas-tight glass syringe with isoflurane. Avoid introducing air bubbles. Connect the syringe and liquid inlet with the supplied tubing and move the pusher forward until the liquid in the connecting tube is visibly just about to enter the vaporizer.
   NOTE: Do not store isoflurane in the syringe when not in use.
2. Anesthetize animals with isoflurane in the induction chamber, with an airflow maintained at approximately 400 - 500 ml/min and the concentration of isoflurane at 3.5%.
   NOTE: The unit will not operate without airflow.
3. Once the animals are asleep, turn the stopcock on the anesthesia unit to route the isoflurane gas to the mask piece. Check that the mask piece is intact to avoid leakage of gas. Airflow can be maintained at 400 ml/min, alternatively decreased to 200 ml/min. Reduce isoflurane concentration to 2.6%.
   NOTE: Anesthetic effect can be increased and/or decreased by adjusting the flow rate.

4. BCG Injections and Recovery

1. Visually confirm and perform tests such as the toe pinch reflex test to ascertain that mice are immobilized/asleep on the isoflurane mask piece before performing injections.
2. Inoculate in the hind footpad, 1 x 10⁶ Colony-forming units of Mycobacterium bovis Bacille Calmette-Guérin (BCG) in 30 µl of phosphate-buffered saline (PBS) using a syringe fitted with a 29 G x ½ inch needle.
   NOTE: The generation of mycobacterial stocks is briefly described in the original description of this procedure, and in substantial detail elsewhere. BCG is also readily available from commercial sources. The authors recommend that BCG be pulse-sonicated or passed through a syringe to disrupt clumps prior to its inoculation into mice. BCG is reiterated here as a microbial stimulus given its employment in the original description of this protocol 3 but the authors certainly encourage the testing of other microbial stimuli.
3. Perform the same procedure described above for the injection of PBS into control mice.
4. Monitor animals after injections to confirm that recovery from anesthesia is successful and that animals can support themselves on the injected, hind footpad.

5. CFSE Injections

1. Preparation of CFSE for Injections
   1. Thaw a vial of 5 mM CFSE stock solution from -80 °C. Dilute the CFSE 10-fold in PBS. Resuspend the solution thoroughly before filling the syringe in preparation for the injection.
2. CFSE Injection and Recovery
   1. Repeat the procedure for anesthetizing animals in section 3. Inject 20 µl of 0.5 mM CFSE in the hind footpad which previously received BCG or PBS.
      NOTE: The injection of CFSE should be performed the day before (24 hr) the planned date for sacrifice.

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.

1. Euthanize the mice. Spray the animal with 70% ethanol. Pin the animal in the dorsal position, on a dissection board.
   NOTE: The authors can recommend euthanasia through cervical dislocation.
2. Carefully make a vertical incision in the hind thigh. Clear muscle and fat using scissors and tweezers to expose and consequently excise the pLN located deep in the fat pouch, in the hollow of the knee.
3. Transfer the pLN to 0.5 ml sterile PBS, on ice.

7. Generation of Single-cell Suspension from pLN

1. Homogenize the pLN through a 70-micron cell strainer placed in a 6-well-plate containing 5 ml PBS or fluorescence-activated cell sorting (FACS) buffer (PBS containing 2% fetal calf serum (FCS), 5 mM ethylenediaminetetraacetic acid (EDTA), and 1 mM sodium azide). Homogenize through the strainer using the back end of a 3 ml syringe.
   NOTE: Sodium azide is toxic and should be handled with caution.
2. Wash the strainer with another 5 ml of PBS or FACS buffer, collect the material in a 15 ml tube, and pellet the cells at 277 x g (1,200 rpm, R = 172 mm) for 5 min, 4 °C. Alternatively, homogenize pLNs directly in microcentrifuge tubes using polypropylene homogenizers.
3. Based on the pellet size, resuspend the pLN suspension in an appropriate volume of PBS or FACS buffer, e.g., 200 - 1,000 µl. Use a counting chamber to quantify the total cell number obtained from each LN suspension. Use trypan blue to exclude dead/dying cells.

8. Cell Staining and Flow Cytometry

1. Transfer 1 - 10 x 10⁶ cells to 5 ml round-bottom polystyrene tubes. Wash with FACS buffer, and pellet by centrifugation at 277 x g (1,200 rpm, R= 172 mm) for 5 min, 4 °C.
2. Discard the supernatant and resuspend the pellet in 50 - 100 µl of antibody cocktail (see Dendritic cell [DC] markers used for surface staining, Materials Table) containing 0.5 - 1 µg of anti-mouse CD16/CD32 (to block unspecific Fc-mediated interactions) in FACS buffer for 30 - 45 min, on ice. Protect samples from light.
3. After incubation, wash cells with FACS buffer and pellet by centrifugation at 277 x g (1,200 rpm, R = 172 mm) for 5 min, 4 °C.
4. Resuspend cells in 50 - 100 µl of FACS buffer. Acquire data on a flow cytometer.

Materials

Name	Company	Catalog Number	Comments
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