Kinetics of White Blood Cell Staining by Intravascular Administration of Rhodamine 6G

Abstract

Rhodamine 6G is a vital dye accumulating in the mitochondria of cells. It is used in intravital fluorescence microscopy for contrast enhancement of white blood cells (WBC), enabling visualization of WBC in the microvasculature even at high center flow velocity. The aim of this study was to examine the kinetics of WBC staining after intravascular administration 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 fluorescence intensity were measured by flow cytometry 5, 15, 30 and 60 min after dye administration. Injection of 0.06-0.2 mg/kg body weight of rhodamine 6G resulted in staining of practically all granulocytes and monocytes over the entire observation period of 60 min. Fluorescence intensity of WBC was adequate to be detected in an experimental setup for intravital fluorescence microscopy in the hamster dorsal skinfold chamber. The degree of WBC staining was different in the species studied, yielding a higher percentage of stained lymphocytes in rats than in mice and hamsters. Staining of lymphocytes declined within 60 min after rhodamine application, the loss of fluorescent label being most pronounced in hamster cells. After 15-30 min, relative fluorescence intensity of stained lymphocytes had decreased considerably, indicating the need for reinjection of the dye or limiting microscopic analysis to approximately 15 min after rhodamine 6G administration. While the intravascular injection of rhodamine 6G results in adequate staining of granulocytes and monocytes, only a fraction of lymphoid cells are stained.

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“…Intravital microscopy of the mouse cerebromicrovasculature was performed as previously described [18]. Briefly, the mice were anesthetized by i.p.…”

“…Mice were injected via the tail vein with 200 l of a 0.05% rhodamine-6G solution: 5 mg of rhodamine-6G were dissolved in 10 ml of 0.9% saline solution. Rhodamine 6G selectively stains mitochondria and achieves 99.99% staining of leukocytes for up to 5 min after injection of the concentration used in this study [18]. Then, leukocytes were examined using a Zeiss Axioplan Calculations were based on external standard readings, and extravasated dye was expressed as g of Evans Blue/mg dried weight of brain tissue.…”

Microvascular blood flow and stasis in transgenic sickle mice: Utility of a dorsal skin fold chamber for intravital microscopy

“…Venules with no observable blood flow were reported as static. For measurement of leukocyte rolling and adhesion, the leukocytes were labeled in vivo with 100 mL of 0.02% rhodamine 6G (Sigma Chemical Co., St. Louis, MO) in sterile saline administered intravenously via tail vein injection [44]. The DSFC was illuminated with a mercury lamp and rhodamine signal-enhancing filters (excitation l ¼ 480-520 nm and emission l ¼ 535-585 nm) were used to view the fluorescence.…”

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“…; Sigma-Aldrich, St. Louis, MO), immediately before microscopic visualization. Rhodamine 6G at the dose used labels leukocytes and platelets, allows detection of the same number of rolling leukocytes as transmitted light, and has no effect on leukocyte kinetics (22). Rhodamine 6G-associated fluorescence was visualized by epi-illumination at 510-560 nm using a 590-nm pore size emission filter (22).…”