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

METHODS 

• Blood from Jugular vein of cattle is taken in a sterile flask having 2.7% EDTA solution at the ratio of 20 : 1
• The blood is centrifuged at 1500 rpm for 30 minutes
• The buffycoat from the above tube is taken out in a seperate centrifuge tube and suspended in 10 ml of Ca+-Mg+ free PBS.
• The tube is centrifuged at 1200 rpm for 10 min.
• The supernatant is taken out and the cell pellet is againg suspended in 10 ml of Ca+-Mg+ free PBS and centrifuged at 1200 rpm for 10 min.
• The cell pellet is suspended in the plain RPMI 1640 medium and 0.1 ml of cell suspension is mixed with 0.9 ml of trypan blue.
• The cell number and the viability are checked in a haemocytometer. 
• Number of cells is counted in atleast two peripheral 16 square chambers and average of two is taken.
• Cell no. = No. of cells x 10⁴    x10x10
• Cells taken trypan blue dye should not be counted as these cells are dead.
• For lymphocyte stimulation test, the viability of cell should be more than 90%.
• Peripheral blood lymphocytes are suspended in the growth medium (RPMI 1640 containing 10% FCS, 0.02% HEPES, antibiotics) at the rate of 2x10 ⁶  / _^ml, Con A 10ug/ ml or PHA 10ug / ml or specific antigen of required amount in triplicate each in 96 wells cell culture plate.
• The TC plate is incubated in a CO2 incubator at 37 ⁰ C in a humidified atmosphere for 72 hr. 
• Each well is pulsed with 1uc of 3H thymidine and incubated further o/n at the above conditions and cells were harvested on a cell harvester.
• The counts are taken in a beta-counter.
•  Stimulation index is calculated as below:

 CPM in stimulated / CPM in unstimulated

• The stimulation index more than 2 is positive.