The overall goal of this procedure is to generate a lymph node fat pad chimera to assess the contribution of adipose tissue to the formation of the lymph nodes stroma. This is accomplished first by isolating lymph nodes from newborn mice and fat pads from day 18.5 mouse embryos. The lymph node is removed from the embryonic fat pad and replaced by the newborn lymph node.
The lymph node fat pad chimera is assembled by reassociating and then culturing one newborn lymph node with one embryonic fat pad in vitro. The chimera is then grafted under the kidney capsule of a host mouse. Three weeks after transplant, the kidney is harvested and the chimera is retrieved.
Ultimately, the contribution of the fat pad derived cells to the lymph node stroma can be assessed by flow cytometric and immunofluorescence microscopic analysis. So we first had the idea for this method when we were searching for a way to assess whether cells from the fat pad we contributing to the formation of the lymph nodes To isolate newborn inguinal lymph nodes. After sectioning the head use a pair of scissors to open the body of the animal from the top of the thoracic region to the bottom of the abdomen.
After carefully removing all the viscera from the abdominal cavity, place the body into a 90 millimeter Petri dish containing RF 10 media. Place the dish in a sterile tissue culture hood and then transfer the body into a new sterile 90 millimeter Petri dish containing fresh RF 10. Then carefully detach the peritoneum from the skin in the inguinal region and locate the inguinal lymph nodes situated at the intersection of three blood vessels in the fat pad.
Carefully remove the lymph nodes, making sure that all the adipose tissue is removed. Then place the lymph nodes into a 50 millimeter Petri dish containing RF 10 media on ice to isolate the inguinal fat pads from day 18.5 embryos. After removing the viscera as just demonstrated, wash the bodies in sterile PBS to eliminate all traces of blood.
Transfer the cleaned bodies to a fresh 90 millimeter Petri dish and then detach the peritoneum, localize the inguinal lymph node and remove it as just demonstrated. Discard the isolated tissue. Take care to remove the entire lymph node to avoid contamination of the fat pad chimera.
Then remove the inguinal fat pad and place it into a 50 millimeter Petri dish containing RF 10 media on ice. To set up the in vitro organ culture system, first cut some Vulcan under wrap into one to 1.5 square centimeter pieces. Then boil the sponges for two hours and the filters for 20 minutes in distilled water and let them dry for several hours in a cell culture hood.
Once the materials are dry, place one sponge each into 50 millimeter Petri dish containing two milliliters of media. Submerge each sponge in the media to wet both sides, and then place one filter per in vitro organ culture system at the liquid air interface. Next, carefully reassociate one embryonic fat pad with one newborn lymph node directly onto the top of each filter.
Transfer the Petri dishes into a rectangular plastic box with water at the bottom and holes in the lid. Tape the lid to the box leaving the holes open. Then place the box in a 37 degree Celsius 5%CO2 cell culture incubator.
Allow the box to equilibrate for two hours and then seal the holes in the lid with tape. Incubate the tissues for at least two days to allow the lymph nodes to attach to the fat pads before transfer under the kidney capsule, three to four weeks after transplantation. Isolate each lymph node fat pad chimera within each kidney and dissect out the lymph nodes.
Then for each lymph node, use a small pair of scissors to make a single incision in the tissue to aid in the enzymatic digestion. Next place one lymph node each into individual 1.5 milliliter einor tubes containing 600 microliters of digestion buffer. Incubate the tubes for 30 minutes at 37 degrees Celsius on an agitating thermal block, pipetting up and down every 10 minutes to help dissociate the tissue.
Then add six microliters of EDTA to the tubes and tritrate the cell suspension a few times to finish the dissociation. The cells can then be stained with the desired antibodies of interest and analyzed by flow cytometry to preserve the enhanced yellow fluorescent protein or EYFP expression. Fix the lymph nodes for three to four hours.
Then for each lymph node, place a single drop of cold OCT compound onto a small piece of aluminum foil. Avoiding air bubbles carefully place one lymph node into the middle of each drop and then place the foil onto dry ice to freeze the tissues. The lymph nodes can then be sectioned, stained, and analyzed by fluorescent microscopy.
P careful isolation of the lymph node allows further analysis of the progeny of EYFP positive adipose-derived cells. Cryo sections and immunofluorescent analysis of the lymph node reveals that EYFP positive adipose-derived cells migrate into the lymph node where they contribute to the GP 38 positive E RTR seven positive lymph node stromal cell network flow Cytometric analysis confirms that an important fraction of the lymph node stromal cells derives from local EYFP positive adipose tissue progenitor cells contributing to 30%of the CD 45 negative G 38 positive CD 31 negative FIBROBLASTIC fraction, and 10%of the CD 45 negative GP 38 negative CD 31 positive blood endothelial cell fraction up to 80%of the CD 45, negative G 38 positive CD 31 negative fibroblastic fraction can be derived from adipose tissue precursor cells demonstrating the crucial role played by adipose tissue in sustaining the growth of the lymph node stroma. So once mastered this technique with a researcher in the field of lymphoid Organogenesis to explore the role of different molecules involved in the of lymphoids trauma.
