1. Immunization
An appropriate adjuvant is composited with microgram or milligram amounts of immunogen and injected at multiple site of the mouse at various times in a repetitious manners. At times the is bled and test for antibodies of aimed specificity is made. Highest concentration of antibodies are established after the mouse is immense with 2-3 individual miniature doses of antigen. When concentration of antibodies are maximum the animal is sacrificed and the spleen is taken. Then it is separated into individual spleenocyte by applying enzyme or instrumental process. Density gradient centrifugation is mostly used to separate the lymphocyte of the spleen from the rest of cells organelles.