Immunostimulatory Agent Evaluation: Lymphoid Tissue Extraction and Injection Route-Dependent Dendritic Cell Activation

The overall goal of this procedure is to familiarize the unique technique required in isolating mediastinal lymph node from mice. The operation was conducted after injecting immunostimulant material on six different routes in mice. In this study, we used a new class of material forming nano-sized structures.

Surfactants consist of hydrophilic head group and hydrophobic tail. Those form spherical micelle or other secondary structure due to microphage separation. We utilized this property to a bio macromolecule that is DNA.

DNA amphiphile, shown here, as regular nucleotides such as A, C, G, T, has hydrophilic blocks while lipid modified neuro-cell nucleobases act as the hydrophobic units. The modified oligo was synthesized by DNA synthesizer in large scale octograms. In equi-media the amphiphilic lipid DNAs spontaneously form nano-sized spherical micelles.

When micelle is formed, the DNA remains single-stranded as an empty carrier with about 10 nanometer in diameter. To equip the carrier with immune-stimulant, it is simple matter of extending the DNA sequence with toll-like receptor nine adjuvant namely eCpG. Lipid DNA and eCpG base here therefore the outer surface of micelle is degraded with CpG sequence.

We then name this Immune-Stimulating Nano particle as the INP. To determine the proper way of INPs for immune response we injected INPs to mice in 64 injection routes and consequently a proper lymph node tissue were analyzed. For more detailed injection instruction please find a JOB article published by Charles River in 2012.

The main advantage of this video is visually showing how to obtain mediastinal lymph node. The video explicitly shows the location of mediastinal lymph node which a lot of people find a difficulty in isolating the organs. All experiments were carried out under the guidelines of the Institutional Animal Care and Use Committee at the Shanghai Public Health Clinical Center, or University of Ulsan College of Medicine.

This part will show the specific location of organs or tissues, which are crucial for immunological analysis. Spleen and two lymph nodes will be isolated. First of all, mice were sacrificed by the carbon dioxide inhalation euthanasia and all efforts were made to minimize suffering as following the guidance of Institutional Animal Care and Use Committee.

From now on let's find the organs and isolate them. First, properly fix the mouse on the operating desk with four pins on the palm of the mouse. Sterilize the dissection area with alcohol.

Dissect the outer cover the bottom of the mouse abdomen with a big-sized scissor. Use forceps to open the both sides of attachment and fix it with pins on the operating table. The location of inguinal lymph node is exposed by finding the conjunction of vessel going down toward the left leg.

The shape of the vessel looks like tilted alphabet letter Y.Uncover the layer with the forceps and pull it from the skin. Cut the peritoneum with smaller scissor to expose internal organs. Move intestine to the right by using forceps then wrap in shaped spleen, which show on the left, upper left side of the abdomen.

Additional attention is required during the procedure since spleen is easy to disrupt. Finally the key point of this video:harvesting mediastinal lymph node. It is located underneath the upper side of the lung, which is hard to see due to its small size and somewhat hidden location.

Carefully expose the area using small scissors and forceps. Cut both sides of the ribs and the diaphragm as well. Clip the cut off ribs above the chest.

During the procedure avoid damaging heart and blood vessels since it might blind the site, finding the translucent mediastinal lymph node. If by accident the blood vessel burst, gently wipe out the blood with gauze. Grab the left side of the lung and softly turn it over to the other side to see underneath the lung.

Pull up the mediastinal lymph node using forceps. After harvesting the mediastinal lymph node, make sure to perfectly remove all the fat and blood surrounding the lymph node since undesired tissue may influence the analysis. Finally, all the isolated organs on a petri dish filled with media.

Total 0.1 times 10 to the sixth cells were analyzed on this flow cytometry. Based on force getter and size getter leukocyte population was gated and that cells were excluded by dark staining. 24 hours after INP injection by 64 injection routes, spleen, inguinal lymph node, and mediastinal lymph node were harvested and analyzed DC activation by flow cytometry.

Based on the strategy of flow cytometry DC population was defined as CD11C positive and lineage negative cells in the live leukocyte. Notably, intranasal injection of INP promoted significant increases in the CD40, 80, 86 expression in the mediastinal lymph node compared to PBS for the control. Therefore, this data suggested that intranasal injection of INP can be used as a mucosal adjuvant for enhancement of immunity in the lung.