Cell experiment:

Malignant cells and normal control cultures are seeded in equal (protein adjusted) cell amounts into 6-well tissue culture plates. The cells are pulsed with 25μCi/mL of 3H-Thymidine and immediately treated with Rhodamine 6G at the fixed concentration of 1 μM for 24h, 48h, 72h or 5 days (120h). Following 24h, 48h, 72h or 5 days, the excessive radioactive material is ished out with PBS. The cell samples are transferred into polystyrene vials containing 4 ml scintillation liquid, and their radioactivity counted in a β-counter. Total cell protein is assessed by Bradford’s assay[2].

Animal experiment:

Mice: C57Bl mice are implanted with B16-F10 melanoma and treated with Rhodamine 6G (1, 0.1, 0.01 μM) at different dosage/time regimens. Viability and proliferation of cultured tumor cells are analyzed[2].

Rhodamine 6G is a rhodamine analog useful in Pgp efflux assays. It can be used in characterizing the kinetics of MRP1- mediated efflux, and as a laser dye and potential mitochondrial probe.

Rhodamine 6G, also known as Rhodamine 590, is widely used as a lasing medium and as a fluorescence tracer. For use in dye lasers, it is dissolved in methanol, ethanol and a variety of other organic solvents. In environmental flow studies, the tracer medium is typically water. In ethanol, the absorption of rhodamine 6G ranges between 440 nm and 570 nm, with the peak at 530 nm. It is thus ideally suited for pumping by frequency doubled Nd:YAG lasers at 532 nm, copper vapor lasers at 511 nm, and argon-ion lasers at 514 nm. The resulting emission spectrum varies from about 510 nm to around 710 nm with the peak at 550 nm depending on the solvent and the dye concentration. However, the laser emission range is much narrower, from around 560 nm to 610 nm with the peak wavelength around 575 nm. Energy conversion efficiencies greater than 50% are achievable. In order to select the optimal solvent and dye concentration, good knowledge of their effects is a pre-requisite. Rhodamine 6G in DMSO shows a distinct behavior exhibiting only 41% of the fluorescence intensity of the methanol case with an 11 nm red-shifted wavelength. Relatively small changes of the fluorescence spectrum are observed for the different solvents; the highest fluorescence intensity is observed for methanol and lowest for DMSO. The shortest peak wavelength is found in methanol (568 nm) and the longest in DMSO (579 nm). Changing the dye concentration provides tunability between 550 nm in the dilute case and 620 nm at high concentration, at which point the fluorescence spectrum indicates the formation of rhodamine 6G aggregates[1]. Rhodamine 6G is a fluorescent dye capable of penetrating a living cell. Upon entering the cell, Rhodamine 6G binds to the inner membranes of mitochondria. Based on these observations, it has been proposed that Rhodamine dyes may be used for producing fluorescent images of mitochondria with low background noise and high resolution. Extremely low concentrations of Rhodamine 6G appear to selectively destroy malignant cells in culture, sparing the normal cell populations[2].

Melanoma-transplanted mice receiving Rhodamine 6G demonstrate prolonged survival, improved clinical parameters, inhibited tumor growth and metastases count, compared to their untreated counterparts. Twice-a-week 10-6M Rhodamine 6G regimen yield the most prominent results[2]. The Rhodamine-6G enters the circulatory system and labels leukocytes. It is possible to monitor changes in the interactions between leukocytes and the endothelium by determining the numbers of rolling and adhering leukocytes as well as the total flux of these cells[3].

[1]. Zehentbauer FM, et al. Fluorescence spectroscopy of Rhodamine 6G: concentration and solvent effects. Spectrochim Acta A Mol Biomol Spectrosc. 2014;121:147-51. [2]. Kutushov M, et al. Low concentrations of Rhodamine 6G selectively destroy tumor cells and improve survival of melanoma transplanted mice. Neoplasma. 2013;60(3):262-73. [3]. Jain RK, et al. Measuring leukocyte-endothelial interactions in mice. Cold Spring Harb Protoc. 2013 Jun 1;2013(6):561-3.