Splantation Procedure

In order to begin, the mouse has to be placed on its back on a Styrofoam board covered in multiple layers of paper towels and one layer of aluminium foil. It is then fixed by the limbs with needles.

Afterwards, the skin is carefully cut without damaging the peritoneum and removed from the front of the body, also fixing it with needles, to gain access to the inner organs.

Once the area is free, the first cells to be isolated are taken from the peritoneal cavity. A cold mix of PBS and BSA is injected with a syringe into the peritoneal cavity from the upper abdominal part. The cells are then dislodged into the liquid by shaking the board and tapping the body of the mouse.

The next step is to cut a hole into the peritoneum and collect as much

The second organ to be extracted is the spleen. In order to do this, the peritoneum has first to be cut and fixed at the sides with the help of the needles that were holding the skin.

Next, the internal organs contained in the abdomen are moved to the left to facilitate the location of the spleen. It is then pulled out carefully with tweezers, using another set of tweezers to help to separate the remaining connective tissue from the organ. The spleen is then placed in a sieve, mashed with the plunger of a syringe and washed through with 10 mL of cold PBS/BSA mixture into a falcon tube. The cells are then prepared.

Then, the thymus is isolated. For this, the sternum is cut and moved from its place. Although the script said to cut at each side of the sternum and to pull it up towards the head [7], in this case, the sternum was cut through the centre and pulled to the sides, as this made it easier to gain access as well as to save time by cutting only once. The thymus is then carefully pulled out with tweezers, paying special attention not to break the heart in order to avoid

This is incubated at room temperature for 15 minutes. Once the incubation time is finished, the tube is filled up to 10 mL with cold PBS/BSA and centrifuged again with the same conditions as the first time.

The supernatant is once more discarded and the pellet is resuspended in 500 μL PBS/BSA.

The cells from the thymus and the peritoneal cavity are not lysed, but instead only spun and resuspended in 500 μL PBS/BSA.

Later, the cells from each organ are counted. In order to do this, a predilution with PBS is made. Both thymus and spleen were diluted by a factor of 1:25, following the script [8]. The bone marrow suspension had a 1:10 dilution for counting, instead of 1:4 as suggested [8], and the peritoneal cavity cells had to be diluted by a factor of 1:2 due to a very high cell number in the undiluted suspension [8].

Afterwards, 10 μL of diluted cells were mixed with 10 μL of Trypan blue and put into a Neubauer chamber for counting.

The last procedure before analysis is FACS staining. Here 4x105 cells are pipetted into a well of a 96-well plate for each organ and filled up to 250 μL with PBS/BSA. The plate is then centrifuged at 300x g for 5 minutes at 4 ºC. The supernatant is discarded and the pellet is resuspended in 20 μL antibody