PREPARATION AND STUDY OF SPLEEN CELLS
Prepare Cell Suspension from Spleen
Remove spleen
1. pin the animal to the dissecting tray.
2. wet the fur with water or alcohol
3. with scissors make a ventral cut along the middle of the abdomen about 7-12 cm
long to expose the viscera
4. locate the spleen on the left side side and cut around the connective tissue to free
the organ being careful not to puncture the capsule
5. place the spleen into a Petri dish with enough cold PBS ( phosphate buffered
saline) to cover the organ
6. keep everything on ice from now on
Remove the cells from the spleen (keep everything as cold as possible)
1. pick up the spleen and place it onto a screen mesh partly immersed in PBS ( you
can use the same pteri dish) – keep the tissue wet with PBS
2. using small scioors mince the spleen on the screen
3. with a syringe plunger push the small fragments of spleen through the tissue .
4. rinse the plunger and screen with a pastuer pipette full of PBS
5. discard the screen and allow the large clumps of tissue to settle in the Petri dish
6. remove the unclumped supernatant with a Pasteur pipette to a 15 ml test tube.
Enrich for WBCs
1. if the tube with the spleen cells contains less than 3 ml of fluid , add PBS to the 3
ml mark
2. allow this tube (covered) to sit on ice for 10 minutes.
3. at the end of this time period, remove the supernatant with a pp (Pasteur pipette)
to a clean 15 ml tt leaving about 0.5 ml of liquid at the bottom of the old tube.
Quantifying Cells : RBCs and WBCs
1. Determine the concentration and total number of cells in your spleen suspension
by using a hemacytometer.
2. Determine the % of RBC and the % of WBC in your suspension.