VMC 221: Veterinary Immunology and Serology (1+1)

PROCEDURE

• Draw 5-6 ml of blood in 250 units of heparin from ear vein in sterile condition.
• Add heparinized blood to a siliconized round bottom tube containing 2 ml of 3% Dextran.
• Keep the tube in a slanting position at 37 ⁰ C for 45 minutes. Then keep vertically for 15 minutes at 37 ⁰ C.
• Take out the leukocyte rich plasma with a Pasteur pipette into a siliconized centrifuge tube and centrifuge at 900 rpm for 5 minutes. Wash the pellet once with HBSS.
• If the pellet contains excess erythrocytes add 4-5 ml of 0.87% ammonium chloride and leave at room temperture for 2-3 minutes and then centrifuge at 900 rpm for 5 minutes.
• Wash the cells twice with HBSS and adjust the final cell suspension to contain 15 x 106 cells per ml.
• Fill the capillary tubes with cell suspension, plug at one end with ‘Sealease’ clay and centrifuge at 500 rpm for 4 minutes.
• Make the Perspex chambers pyrogen-free by immersing them in 5% hydrogen peroxide and then drying.
• Cut the capillaries at the cell fluid interface and affix with silicon wax in 1.5 ml Perspex chambers.
• Fill the chambers immediately with HBSS enriched with 5% new-born calf serum.
• Fill few chambers with enriched HBSS to which antigen has been added. (The dilution of antigen to be used in the test should have been tested earlier for its toxicity to leucocytes of normal animals).
• Close the chambers immediately with cover slips taking care not to leave any air bubble into the chambers.
• Incubate the chambers in a humid chamber at 37 ⁰ C for 20 hours and record the area of migration in control as well as antigen chambers on a centimeter graph sheet with the aid of a Camera Lucida and calculate the area of migration.