The protocol described here utilizes a photolabeling approach in newborn mice to specifically identify immune cells that emigrate from the colon to extra-intestinal sites. This strategy will be useful to study host-microbiome interactions in early life.
This method can help answer key questions in the mucosal immunology field regarding neonatal host immune microbiome interactions such as do cells carry microbial messages, and if so which cells are they? The main advantage of this technique is that it non-specifically an irreversibly labels colon resident cells in infant mice, enabling us to determine their trafficking patterns to extra-intestinal sites. To assemble the laser, connect the light-emitting diode, or LED to the LED driver via the four-pin M8 connector cable of the LED and carefully tighten the screw to fasten the connection.
Hold the connector opening of the LED up to the light to ensure that there are no visible obstructions. Insert the optogenetics patch cable into the opening. Insert the cannula into either end of the connector sleeve.
Then attach the other end of the connector sleeve to the end of the patch cable, connecting the fiberoptic cannula to the fiberoptic patch cable. Use the continuous adjustment knob to set the LED driver to the continuous wave mode at zero, and the current limit set to 1.2 amps. When the laser is ready, plug the LED driver into a power source in a dark location.
Use one hand to gently scruff the back of an anesthetized day 10 to 12 postnatal mouse with the index finger and thumb. Turn over the animal to expose the abdomen, and secure the tail between the ring and little fingers. Use the other hand to remove the protective cap from the cannula, and gently insert the fiberoptic cannula through the anus, into the colon, so that all five millimeters of the optical fiber are inside the mouse, maintaining the long axis of the cannula parallel to the mouse's midline.
The design of the cannula including its flexibility, diameter, and length is critical to the success of this procedure. The neonatal pup is very fragile and great care must be taken to avoid perforating the colon. Wearing safety goggles, increase the laser current to the maximum value and confirm the presence of UV light by looking at the translucent abdomen of the animal.
Withdraw the cannula approximately one millimeter every 30 seconds to maximize the luminal area exposed to the light. After two minutes total of UV light exposure, slowly remove the cannula from the mouse and turn off the laser. After recovering from anesthesia, return the mouse to the home cage.
To harvest the intestinal lymphocytes, use clean forceps and scissors to open the abdomen, and reflect the intestines to one side to expose the colon. To remove the whole colon, first transect the tissue just distal to the cecum, and firmly grasping the colon with forceps, use scissors to cut through the urethra to the rectum. Then cut the colon as close to the anus as possible, and remove the entire colon from the abdominal cavity.
Use forceps to push the contents out of the colon onto a dry paper towel. And transfer the clean colon onto the lid of a tissue culture dish. Use razor blades to finely chop the sample, and transfer the resulting paste into a cell strainer set in a Petri dish containing 10 milliliters of cold Hank's Balanced Buffer Solution supplemented with calf serum, or HBSS-CS.
Add a clean eight millimeter long magnetic stir bar to the strainer and place the dish on a nine-position magnetic stirrer plate with a magnetic stir bar speed set to 800 RPM. After a five-minute incubation at room temperature, discard the buffer, and repeat the wash with 10 milliliters of HBSS-CS. Dissociate the epithelial cells with 10 milliliters of pre-warmed epithelial strip buffer and stir the cells at 800 RPM for 30 minutes in a 37-degree Celsius incubator.
At the end of the incubation, transfer the enriched intestinal epithelial fraction into a 15 milliliter tube for centrifugation. And re-suspend the pellet in one milliliter of FACS buffer on ice. Next, wash the remaining lamina propria tissue in the strainer two times with HBSS-CS as just demonstrated, followed by two incubations in 10 milliliters of wash buffer supplemented with EDTA for 15 minutes at 37 degrees Celsius with stirring.
Then wash the sample two more times with HBSS-CS, followed by a third incubation in 10 milliliters of EDTA-free wash buffer for 10 minutes at room temperature without stirring. At the end of the incubation wash the sample two more times with HBSS-CS, and digest the remaining lamina propria tissue in the cell strainer with 10 more milliliters of EDTA-wash buffer supplemented with Dnase I and collagenase with stirring at 800 RPM for 45 minutes at 37 degrees Celsius. At the end of the digestion filter the enzyme solution through a new strainer into a 50 milliliter conical tube and neutralize the collagenase activity with 20 milliliters of cold wash medium.
Collect the lamina propria cells by centrifugation. And re-suspend the lamina propria lymphocyte pellet in one milliliter of FACS buffer. Then combine the intestinal epithelial lymphocytes and the lamina propria lymphocyte fractions, and stain the cells with the appropriate fluorescents conjugated antibodies of interest for flow cytometric analysis.
A 30-second laser light exposure delivers a maximal photo conversion of colon cells with a minimal cytotoxicity. No fluorescents activated cells are detected in the colons of unlasered mice, while 405 nanometers of light exposure results in distinct dendra-r positive cell populations in both the CD45 negative and CD45 positive cell compartments. Of note, no background labeling is observed in the spleen, suggesting that the intracolonic laser beam does not penetrate enough to photolabel cells in extraintestinal sites.
In this representative experiment, the migration of photoconverted intestinal cell migration from the colon to the spleen was evaluated with only CD4 positive cells identified three to five days after laser light exposure, consistent with the theory that CD45 negative cells within the colon are primarily epithelial and structural cells. Once mastered, the light exposure steps can be performed in approximately five minutes per mouse, while the entire sample collection and analysis takes about nine hours to be completed. After watching this video you should have a good understanding of how to label cells in the colons of newborn mice and to use flow cytometry to identify the labeled cells in the spleen after their migration.
Don't forget that working with ultraviolet light and animal anesthetics can be extremely hazardous. Precautions such as wearing safety goggles and using an appropriate scavenging system should always be taken while working with these materials.
