Phagocytosis

Phagocytosis Protocol and Examination

In order to study the locations and functions of the diverse and numerous structures compromising the immune system, focusing on the cells and lymphoid organs of the immune response. In addition, to the process of phagocytosis can be studies, through visualizing and analyzing the phagocytic function of the reticuloendothelial system by mouse macrophages.

Collection, preparation, and visualization of macrophages isolated from the peritoneal cavity of a normal and immunized mouse.

Administration of 0.5 mL of 2% washed foreign (e.g. chicken) erythrocytes with saline is to be administered by intraperitoneal injection into a normal mouse. A negative control can be included in this experiment, which is administered as above and with equal amount of fluid, however, consisting of only saline solution and no foreign erythrocytes. Labeling of the injected mouse is done in order to prevent any mix-up. All injections are to be performed above the lower right quadrant of the abdomen, piercing the peritoneum which lines the peritoneal cavity. Mice are subsequently harvested after 1 week. Both mice are sacrificed by cervical dislocation, followed by the removal of abdominal skin to reveal the peritoneum, which is a sterile body cavity enclosing the abdominal organs, such as the intestines. 3.5 mL of heparinized tissue culture medium containing 2% BSA (bovine serum albumin) is then injected into the peritoneal cavity, with 30 seconds of massaging the belly to allow for the redistribution of the fluid. This is then followed by injection of 3 mL o fluid. BSA is used to prevent any leakage of proteins caused by interstitial fluid dilution. Preventing osmotic pressure changes within the peritoneal cavity subsequently. The anticoagulant properties of heparin are used to prevent any formation of blood clots, through platelet and fibrin formation, from any punctured vessels that would lead to the trapping and loss of leukocytes in the formed meshes of fibrin. Recovered peritoneal fluid from each mouse can then be transferred into individual separate glass petri dishes each with 2 cover glasses. One drop of 2% foreign blood (e.g. chicken) is then added to each dish. Incubation at 37 degrees then commences. Cover glasses are then removed after 5 and 30 minute intervals, followed by fixation and staining. This was done with Wright's stain method. Immersion of the cover glasses in methanol fixes the cells to the glass. Staining with Wright's dye for microscopical examination with oil immersion will allow you to see the cells of the immune system.

Location of the major lymphoid organs in the mouse.

The sacrificed mice are further dissected with the use of surgical scissors and forceps to open the abdomen, to visualize the major lymphoid organs' site of residence in the murine body, as well as their structure. The spleen, thymus, Peyer's patches and peritoneal lymph nodes are particularly examined.

Collection, preparation, and visualization of human blood cells.

Human blood was prepared, through the use of a sterile lancet on an individuals fingertips. A drop of blood is placed on a microscope slide, done in duplicates, spread evemly and prepared using the Wright's stain and the method mentioned previsouly to visualize the murine macrophages. Observations are made using the oil immersion lens.

Examination of Phagocytosis

Under 40X magnification you will be able to observe the murine macrophages, the chicken erythrocytes and any phagocytosis occurring, if present. The process of phagocytosis is seen as an interaction (engulfment) with the externally applied, foreign, chicken erythrocytes. Phagocytosis is easily observed on a 30 minute slide, with 1/3 of all chicken RBCs, from the immunized mice and also with a lesser abundance in the 5 minutes slide, with 1/10 the chicken RBCs. The 5 minute slide from the control mouse should show no phagocytosis, while the 30 minute slide displayed 1/10 the phagocytosis of the chicken RBCs, similar to the 5 minute immunized mouse slide value for phagocytosis.

The staining procedure with Wright's stain. produces red colored chicken erythrocytes with a yellowish-like nucleus and bluish-purple macrophages. The slides consist of red ellipsoidal and nucleated chicken erythrocytes and blue macrophages surrounding and engulfing the chicken erythrocytes.

You will be able to locate and characterize all of the major lymphoid organs. The thymus is easily located by opening and separating the mouse's surrounding rib cage and looking just above the heart. The thymus is characterized as a small, twin-lobed white organ. To locate the spleen, Peyer's patches and peritoneal lymph nodes, the peritoneum was first cut open. In the left quadrant in the exposed abdomen was the stomach, an inflated pouch, underneath of which was the spleen. The spleen is oval-shaped, thin and blood-red in color. Observing the surface mucosa of the small intestine, which is attached directly to the stomach, revealed Peyer's patches. The Peyer's patches are small, yellow, elevated patches. Beneath the intestines are tissues, which anchor the intestines within the abdominal cavity. These anchoring tissues contain the peritoneal lymph nodes which are visible as three large white nodes. The majority of the identity of the organs is further validated by textbook descriptions.

References:

1. Janeway, C.A., Travers P., Walport M., and Capra J.D. 1999. Immunobiology: The immune system in health and disease. Garland Publishing, 4th ed., New York, USA, pages 1-40, 2.5-2.22, 3.1-3.12

2. DeFranco, A.I. 1987. Molecular aspects of B-lymphocyte activation. Annu. Rev. Cell Biol. 3:143-178

3. MacLennan, I.C.M. 1994. Germinal centers. Annu. Rev. Immunol. 3:117-139

4. Ravetch, J.V. and Kinet, J. 1993. Fc receptors. Annu. Rev. Immunol. 9:457-492