KINETICS OF WHITE BLOOD-CELL STAINING BY INTRAVASCULAR ADMINISTRATIONOF RHODAMINE 6G

Rhodamine 6G is a vital dye accumulating in the mitochondria of cells. It is used in intravital fluorescence microscopy for contrast enhance ment of white blood cells (WBC), enabling visualization of WBC in the microvasculature even at high center flow velocity. The aim of this st udy was to examine the kinetics of WBC staining after intravascular ad ministration of rhodamine 6G in Lewis rats, Syrian golden hamsters and BALB/c mice. For this purpose, WBC were isolated from whole blood and the percentage of cells stained positively as well as their fluoresce nce intensity were measured by flow cytometry 5, 15, 30 and 60 min aft er dye administration. Injection of 0.06-0.2 mg/kg body weight of rhod amine 6G resulted in staining of practically all granulocytes and mono cytes over the entire observation period of 60 min. Fluorescence inten sity of WBC was adequate to be detected in an experimental setup for i ntravital fluorescence microscopy in the hamster dorsal skinfold chamb er. The degree of WBC staining was different in the species studied, y ielding a higher percentage of stained lymphocytes in rats than in mic e and hamsters. Staining of lymphocytes declined within 60 min after r hodamine application, the loss of fluorescent label being most pronoun ced in hamster cells. After 15-30 min, relative fluorescence intensity of stained lymphocytes had decreased considerably, indicating the nee d for reinjection of the dye or limiting microscopic analysis to appro ximately 15 min after rhodamine 6G administration. While the intravasc ular injection of rhodamine 6G results in adequate staining of granulo cytes and monocytes, only a fraction of lymphoid cells are stained.