Method subcutaneous (SC) administration of antigen to rabbits

The rabbit should be restrained in the normal manner. With your fingers, lift the skin to make a “tent”. Disinfect the injection site and insert needle into the subcutaneous tissue. Aspirate prior to making the injection. Proper placement should yield no aspirate. Inject. Most common injection site is the loose skin around the neck and shoulder area.

12. What are the organs of the immune system are central and peripheral? What lymphoid organs contain mature T- and B-lymphocytes?

The organs of the immune system are positioned throughout the body. They are called lymphoid organs because they are home to lymphocytes, small white bloodcells that are the key players in the immune system.

There are two groups of immune system organs.

· Primary (central)--organs where immature lymphocytes develop

· Thymus

· Bone marrow

· Secondary (peripheral)--tissues where antigen is localized so that it can be effectively exposed to mature lymphocytes

· Lymph nodes

· Appendix

· Peyer's Patches (of GI tract)

· Tonsils

· Adenoids

· Spleen

· MALT (Mucosal-Associated Lymphoid Tissue)

· GALT (Gut-Associated Lymphoid Tissue)

· BALT (Bronchial/Tracheal-Associated Lymphoid Tissue)

· NALT (Nose-Associated Lymphoid Tissue)

· VALT (Vulvovaginal-Associated Lymphoid Tissue)

13. From which bodies receive T- and B-lymphocytes? The method of isolation of B-lymphocytes from the bone marrow of mice.

· Euthanize mice using the institution's animal care committee-approved protocol and spray the animal surface with 70% ethanol.

· Make an incision of the skin in the mid-abdomen and remove the skin from the distal part of the mouse including the skin covering the lower extremities.

· Cut off the muscles from the lower extremities using scissors and carefully dislocate the acetabulum from the hip joint, while avoiding breaking the femur head.

· Remove the remaining muscles from the femur and tibia using a scalpel and scissors and separate the femur from the tibia at the knee joint exercising care to not break the bone ends. Place the bones in a Petri dish containing ice-cold RPMI 1640 1X supplemented with 10% FBS and 1% Penicillin/streptomycin.

· Proceed to the following steps under a tissue culture hood. Take extra precaution to maintain strict sterile techniques to avoid neutrophil activation.

· Rinse each bone with 70% ethanol (within a Petri dish) followed by three subsequent washes in ice-cold sterile PBS (within Petri dishes) to rinse off the ethanol from the surface of the bones.

· Inside a clean sterile Petri dish, cut off the epiphyses of the bones and keep them aside.

· Use a 25-gauge needle and a 12 cc syringe filled with RPMI supplemented with 10% FBS and 2 mM EDTA, and flush the bone marrow cells from both ends of the bone shafts onto a 50 ml screw top Falcon tube fitted with a 100 μm filter. In order to efficiently remove all cells, scrape the inner surface of the bones using the 25-gauge needle.

NOTE: Blanching of bones indicates that the cells have been sufficiently scraped.

NOTE: Use approximately 10 ml of media to flush a femur/tibia pair. Adding EDTA to the medium is essential to prevent clumping of the cells.

· Cut the bone epiphyses in small 0.5-1 mm 3 pieces with a scalpel and smash them through the 100 μm filter using the back end of a 2.5 ml Eppendorf Combitip Plus Biopur pipette tip.

· Centrifuge at 1,400 rpm for 7 min at 4 °C.

· Lyse the red blood cells by resuspending the cell pellet in 20 ml of 0.2% NaCl for approximately 20 sec followed by addition of 20 ml of 1.6% NaCl. (Critical: Do not exceed 20-30 sec of hypotonic lysis to avoid bone marrow cell death. The use of hypotonic NaCl for lysis is recommended over ACK lysing buffer because the latter has the potential to activate the neutrophils).

· Centrifuge for 7 min at 1,400 rpm at 4 °C to collect the cells.

· Wash cells with RPMI 1640 1X supplemented with 10% FBS and 2 mM EDTA and centrifuge as in step 1.12.

· The yield of bone marrow cells using this method is approximately 60-80 million per uninfected 8-12 week-old C57BL/6 mouse.

14. The method of isolation of the spleen cells (splenocytes) in mice.

1. Place the collected Iscoves-Minus Media solution into a 15 ml centrifuge tube.

2. Allow the larger particles to settle to the bottom of the tube. Carefully transfer the supernatant to a new 15 ml centrifuge tube.

3. Centrifuge the supernatant to pellet the red blood cells (RBC) and splenocytes using a clinical centrifuge (or equivalent) at approximately 1,000 X g.

4. Transfer the supernatant to a new 15 ml tube and centrifuge as in Step #3 (above).

5. Lyse the RBC using Tris-NH4Cl Working Solution (1 ml of Tris-NH4Cl per 0.1 ml of packed cells) by gently rocking to resuspend the cell pellet. Incubate for 2 min at room temperature.

6. Underlay (see Hint #1) the RBC lysis solution with 200 μl of Fetal Calf Serum (FCS) and centrifuge to pellet the cells at room temperature using a clinical centrifuge (or equivalent) at approximately 1,000 X g for 10 min.

7. Discard the supernatant and not more than 5 ml of Iscoves-Minus Media solution. Gently resuspend the cell pellet. Centrifuge to pellet the cells using a clinical centrifuge (or equivalent) at approximately 1,000 X g. Repeat washing the cell pellet two more times.

8. Discard the supernatant and resuspend the cells in 3 ml of Iscoves-Minus Media solution.

9. Dilute the cell solution in AKC Lysis Buffer (see Hint #2).

10. Count the cells using a hemacytometer (see protocol on Cell Counting Using a Hemacytometer).

15. Procedure for the preparation of peritoneal macrophage in mice.

Isolation of peritoneal macrophages in mice.

1. Euthanize the mouse, spray it with 70% ethanol and mount it on the styrofoam block on its back.

2. Using a scissors and forceps cut the outer skin of the peritoneum and gently pull it back to expose the inner skin lining the peritoneal cavity.

3. Inject 5 ml of ice cold PBS (with 3% FCS) into the peritoneal cavity using a 27g needle. Push the needle slowly in the peritoneum being careful not to puncture any organs.

4. After injection, gently massage the peritoneum to dislodge any attached cells into the PBS solution.

5. Insert a 25 g needle, bevel up, attached to a 5 ml syringe in the peritoneum and collect the fluid while moving the tip of the needle gently to avoid clogging by the fat tissue or other organs. Collect as much fluid as possible and deposit the collected cell suspension in tubes kept on ice after removing the needle from the syringe.Optional: Repeat step 4-6

6. Make an incision in the inner skin of the peritoneum and while holding up the skin with a forceps use a plastic Pasteur pipette to collect the remaining fluid from the cavity.

7. If in step 6 or 7 visible blood contamination is detected then the contaminated sample should be discarded.

8. Spin the collected cell suspension at 1500 RPM for 8 minutes, discard the supernatant and resuspend the cells in desired media or PBS for counting.

16. What is the reaction to form rosettes with sheep red blood cells?

17. What methods are used for fractionation of whey proteins?

18. Principles chromatographic analysis of immunoglobulins.

19. Principles of electrophoretic analysis of immunoglobulins.

20. What immunoblotting, for what purpose it is used?

Western Blotting (also called immunoblotting) is a technique used for analysis of individual proteins in a protein mixture (e.g. a cell lysate). In Western blotting (immunoblotting) the protein mixture is applied to a gel electrophoresis in a carrier matrix (SDS-PAGE, native PAGE, isoelectric focusing, 2D gel electrophoresis, etc.) to sort the proteins by size, charge, or other differences in individual protein bands. The separated protein bands are then transferred to a carrier membrane (e.g. nitrocellulose, nylon or PVDF). This process is called blotting. The proteins adhere to the membrane in the same pattern as they have been separated due to interactions of charges. The proteins on this immunoblot are then accessible for antibody binding for detection.

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