Fluorescence polarization assay of mitogenic response of lymphocytes

Abstract

Fluorescence polarization measurement was applied to detect the impaired Con A-induced mitogenic responses in tumor-bearing mice. Spleen lymphocytes from normal C3H/HeN mice and from those inoculated 1×106 Ehrlich ascites tumor cells were treated with Con A and examined for their fluorescence polarization changes (P-value) using fluorescein diacetate and a polarization spectrofluorometer. Murine spleen cells when stimulated by 5μg/ml of Con A at 37°C for 45 min showed a high polarization change with a reduction rate of p-value, 0.79±0.03 (n=33). This Con A-induced polarization change of spleen cells was almost completely inhibited by α-methylmannoside, a competitive inhibitor of Con A-binding to the cell surface. Spleen lymphocytes from tumor-bearing mice showed much less polarization changes with a reduction rate of P-value, 0.91±0.04 (n=33). This low fluorescence polarization change appeared as early as 3 days after tumor inoculation. Impairement of mitogenic responses as revealed by 3H-thymidine uptake in tumor-bearing mice also appeared 3 days after tumor inoculation. These results indicate that the fluorescence polarization measurement may be a valuable tools for the detection of impaired mitogenic responses of lymphocytes in tumor-bearing mice.