We describe here a protocol for isolating myeloid cells from mouse skin and draining lymph node following intradermal injection of Plasmodium sporozoites. Flow cytometry of collected cells provides a reliable assay to characterize the skin and draining lymph node inflammatory response to the parasite.
The overall goal of this set of procedures is to immunize mice with radiation attenuated plasmodium parasites. To facilitate the analysis of myeloid cell recruitment in the mouse skin and draining lymph nodes. This method can help answer key questions in the malaria field, such as what are the cellular actors involved in the innate immunoresponse to live attenuated parasites.
Between 18-25 days after the infections blood meal, collect and cold anesthetize infected mosquitoes in a 15 milliliter tube on ice. Next, carefully invert the tube into a Petri dish on ice and expose the insects to a 12 kilorad dose of gamma or X-irradiation. Collect the irradiated mosquito salivary glands in 15 microliters of DPBS on ice.
And then, gently crush the glands to release the sporozoites. The injection of a highly concentrated parasite suspension in a small volume is critical, so it is important to harvest a sufficient number of salivary glands from well-infected mosquitoes as evidenced by robust expression of green fluorescence. Re-suspend the parasite suspension prior to filtration through a 35 micrometer strainer adapted to a 1.5 milliliter microcentrifuge tube.
Using a microscope counting chamber, count the total number of isolated sporozoites and adjust the parasite suspension to an 8.3 times ten to the fourth sporozoites per microliter concentration in cold DPBS on ice. Before injecting the sporozoites, confirm the proper level of sedation of the experimental animal by toe pinch and apply eye ointment to prevent corneal drying. Then, working quickly, use clear tape to gently fix the ventral side of the ear pinna under a stereo microscope and load a ten microliter syringe, equipped with a 35 gauge needle, with 0.6 microliters of resuspended parasites.
Next, carefully insert the needle, bevel side up, beneath the epidermis on the dorsal side of the ear and inject 0.15 microliters of sporozoites into the site. A characteristic papule will be observed at the injection site. After injecting the dermis three more times, as just demonstrated, carefully remove the tape and monitor the mouse until it is fully recovered.
At the desired time point following sporozoite inoculation, sacrifice the mouse and start the procedure immediately. Make sure the mouse demonstrates signs of clinical death. Disinfect the ear and the corresponding neck region with 70 percent ethanol.
Next, using sterile scissors and forceps, make an incision in the jugulo-carotidian area. Then stretch the incision with two forceps to expose the grey-looking draining lymph node. Carefully pinch and remove the connective tissue directly above the lymph node and extract it with a second pair of forceps, placed underneath it.
Transfer the lymph node into a 70 micron strainer in one well of a six-well tissue culture plate, containing 4.5 milliliters of standard medium, supplemented with collagenase and DNase. Next, harvest the entire inoculated ear and use two forceps to pinch and separate the ear into the dorsal and ventral aspects. Then place each side of the ear into a well of a second six-well plate, containing 4.5 milliliters per well of medium supplemented with digestive enzymes, taking care that each tissue is completely immersed in the medium.
To isolate the immune cells from the lymph nodes, incubate the plate of lymphoid tissue at 37 degrees Celsius and five percent CO2 under gentle agitation. After 15 minutes, add ten millimolar EDTA to each well and place the plate on ice. Next, for each lymph node, use the end of a new five millimeter syringe plunger and press the lymph node against the strainer using circular motions to dissociate the tissue.
When only white connective tissue is left, rinse the strainer two times with 500 microliters of DPBS containing FBS and EDTA and transfer the wash to a 15 milliliter conical tube. Then, wash the well two more times with 500 microliters of the supplemented DPBS and transfer the wash to the tube placed on ice. To isolate the immune cells from the ear, incubate the ear leaflets at 37 degrees Celsius and five percent CO2 with gentle agitation.
After 20 minutes, cut the tissues into smaller pieces to facilitate their digestion and return the samples to the incubator. After 40 minutes, stop the digestion with ten millimolar EDTA and place the plate on ice. Using a one milliliter pipette with a cut tip, transfer the entire contents of each well into new wells containing 70 micron strainers.
And use a five milliliter syringe plunger to dissociate the ear tissue fragments with circular motions as just demonstrated. When only white connective tissue and hair remain, wash the strainers and wells with DPBS plus FBS and EDTA as just demonstrated, pooling the washes into a 50 milliliter tube on ice. Finally, wash the well twice with supplemented DPBS and transfer the wash to the tube placed on ice.
In this video, the migration of radiation attenuated sporozoites into the mouse skin 15 minutes after intradermal injection of the parasites can be seen. 24 hours after intradermal sporozoite injection, total cells are isolated from the skin and draining lymph node. As observed on this figure, the frequency of myeloid cells in the skin and draining lymph node is robustly increased compared to naive mice.
Once mastered, the first steps of the protocol for mosquito irradiation to sporozoite injection can be completed in four hours. While attempting this procedure, it is important to remember that sporozoite isolated from mosquito salivary glands rapidly lose their infectivity. So they must be injected as quickly as possible.
After watching this video, you should have a good understanding of how to isolate myeloid cells from the mouse skin and draining lymph node to explore the immunoresponse to intradermal injection of live attenuated plasmodium parasites.
