Abstract
Determination of peripheral lymphocyte subset levels becomes important method for analyzing immune function in nonhuman primates. However, It has been well-known that the separation of peripheral lymphocytes by density gradient centrifuge is relatively difficult in nonhuman primates, because the gravity of red blood cells (RBC) varies among individuals. The purpose of this paper is to compare the major lymphocyte subset levels, the recovery rate of lymphocyte and the contamination of RBC in lymphocyte fraction among four different methods of lymphocyte separation, Method-1: conventional density gradient centrifuge using Ficoll, Method-2: density gradient centrifuge after sedimentation of RBC using 5% dextran, Method-3: whole blood method A; hemolysis before staining lymphocyte, and Method-4: whole blood method B; hemolysis after staining lymphocytes. The lymphocyte recovery rate as well as RBC contamination were also compared between two different hemolysis methods, 1) hemolysis with distilled water and 2) hemolysis with NH4Cl solution. The results obtained are as follows.
I. There was no difference in lymphocyte subset levels between Method-1 and Method-3, indicating that loss of specific lymphocyte subset(s) did not occur during density gradient centrifuge in method-1. The density gradient centrifugal method using Ficoll (d=1.077) was able to seem to adapt for rhesus monkeys.
II. CD16-positive lymphocytes could not be detected by method-4. It might be necessary to wash cells to remove IgG in sample before staining with anti-CD16, Leu-11a monoclonal antibody in the case of applying Method-4 to analyzing CD16-positive NK cells.
III. Method-2 was effective to remove RBC from lymphocyte fraction, but the lymphocyte recovery rate was significantly lower as compared to Method-1.
VI. The lymphocyte recovery rate was significantly higher when contaminated RBC were removed by NH4Cl solution as compared when RBC were removed by distilled water.
