Mitochondria (binds to inner mitochondrial membranes, inhibiting oxidative phosphorylation). [3]

1. Creating the workable solution for Rhodamine 6G 1.1 Get the stock solution ready. To get a 5 mM stock solution, dilute 1 milligram of Rhodamine 6G with 525 μL of anhydrous DMSO. 1.2 Creating a functional solution To prepare a 1-20 μM Rhodamine 6G working solution, use either PBS dilution stock solution or hot serum-free cell culture medium. Note: Please prepare Rhodamine 6G working solution for usage by adjusting its concentration to suit the current circumstances. 2. Staining suspension cells with cells 2.1 Centrifuge the cells, add PBS, and wash twice for five minutes each time. The density of cells is 1×106/mL. 2.2 Add 1 milliliter of Rhodamine 6G working solution, and let it sit for 30 to 60 minutes at room temperature. 2.3 After centrifuging for three to four minutes at 400 g, remove the supernatant. 2.4 After adding PBS, wash the cells twice for five minutes each. 2.5 Re-suspend the cells in 1 milliliter of PBS or serum-free media, and use a flow cytometer or fluorescence microscope to observe. 3. Staining adherent cells in cells 3.1 Use sterile coverslips to cultivate adherent cells. 3.2 Aspirate extra culture medium after removing the coverslip from the medium. 3.3 Cover the cells fully with 100 μL of the dye working solution, shake gently, and allow incubate for five to thirty minutes. 3.4 Aspirate the dye working solution, rinse with culture medium two or three times for five minutes each, and use a flow cytometer or fluorescence microscope to monitor.

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Rhodamine-6G, at concentrations ranging from 10⁻¹² M to 10⁻² M, time- and dose-dependently inhibited the viability and proliferation of various tumor cell lines (B16-F10 murine melanoma, MCF-7 breast cancer, HCT-116 colon cancer, ACHN renal carcinoma, SW-620 colon cancer, and primary murine melanoma cells). Inhibition of ³H-Thymidine incorporation (a measure of proliferation) reached up to 90% in treated tumor cells. [1]
At a fixed concentration of 10⁻⁶ M, Rhodamine-6G treatment for 5 days resulted in the survival of only 15% to 17% of various malignant cells. The most prominent inhibition was observed in B16-F10 murine melanoma cells. [1]
Microscopic examination of tumor cell lines (B16-F10, ACHN, HCT) treated with 10⁻⁶ M Rhodamine-6G for 72 hours revealed massive cell disruption, absence of monolayer confluence, significantly decreased cell count, and visible necrotic, autophagic, and/or apoptotic cells. [1]
In contrast, the same concentration of Rhodamine-6G (10⁻⁶ M) had a negligible effect on normal control cell cultures (human PBMC, HUVEC, rat renal epithelial cells, murine renal mesangial cells, and renal embryonic cells), with 90% to 95% of these cells remaining unaffected after 5 days of treatment. Inhibition of proliferation in normal cells hardly exceeded 15-17%. [1]

Comparing Rhodamine 6G-treated melanoma transplanted mice to their untreated counterparts, the former show superior clinical parameters, longer survival times, and decreased tumor growth and metastasis counts. The most notable results are obtained with a twice-weekly 10-6M Rhodamine 6G regimen[2]. -6G identifies leukocytes when it enters the circulation system. The amount of rolling and adhering leukocytes as well as the overall flux of these cells can be used to track changes in the interactions between leukocytes and the endothelium[3].

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In C57Bl mice transplanted with B16-F10 melanoma, subcutaneous injections of Rhodamine-6G (10⁻⁶ M, 10⁻⁷ M, or 10⁻⁸ M) twice a week significantly suppressed tumor growth. At the endpoint (death of the last untreated control animal), tumors in mice treated with 10⁻⁶ M or 10⁻⁷ M Rhodamine-6G were approximately 200 ± 8 mm³, compared to 320 ± 10 mm³ in untreated controls (p < 0.001). [1]
Treatment with Rhodamine-6G (10⁻⁶ M twice a week) resulted in 40% of tumor-bearing mice surviving at day 31 post-implantation, whereas all untreated controls were dead within 3 weeks. [1]
Mice treated with Rhodamine-6G (10⁻⁶ M twice a week) showed improved clinical parameters, including better fur appearance, normal mobility, and normalized appetite, thirst, urination, and defecation, closely resembling healthy controls. [1]
Rhodamine-6G treatment significantly reduced metastatic dissemination. Animals treated with 10⁻⁶ M Rhodamine-6G twice a week exhibited no visible intra-abdominal metastases, while untreated controls showed extensive metastases on lungs, livers, kidneys, and duodenum. [1]
Preventive treatment (starting 7 days before tumor implantation) with 10⁻⁶ M Rhodamine-6G twice a week resulted in the most significant inhibition of tumor growth compared to treatment started at or after tumor implantation. [1]

Proliferation/Survival Assay (³H-Thymidine Incorporation): Cells were seeded at 2 × 10⁶ cells per well in 6-well plates and cultured for 24 hours. They were then exposed to varying concentrations of Rhodamine-6G (10⁻¹² M to 10⁻² M) and simultaneously pulsed with 25 μCi/mL of ³H-Thymidine. After 48 hours, cells were washed with PBS to remove excess radioactive material, transferred to vials with scintillation liquid, and radioactivity was measured in a β-counter. Results were calculated as mean counts per minute (CPM) per mg of cell protein, as assessed by Bradford's assay. [1]
Time-Course Experiment: Malignant and normal cells were treated with a fixed concentration of 10⁻⁶ M Rhodamine-6G and pulsed with ³H-Thymidine. Cells were harvested and counted at 24h, 48h, 72h, and 120h (5 days) following the same procedure as above to assess proliferation over time. [1]
Morphological Analysis: Cell cultures were examined and photographed under a microscope to assess monolayer confluence, cell count, and signs of cell death (necrosis, apoptosis) following treatment with Rhodamine-6G. [1]

Three-month-old male C57Bl mice (25 ± 5 g) were used. B16-F10 melanoma cells (1.5 × 10⁶ cells in 0.2 mL PBS) were implanted subcutaneously. [1]
For dose-finding, tumor-bearing mice received subcutaneous injections of Rhodamine-6G (0.3 mL bolus) once a week at concentrations ranging from 10⁻¹⁰ M to 10⁻² M. Based on results, concentrations of 10⁻⁶ M, 10⁻⁷ M, and 10⁻⁸ M were selected for further study. [1]
For frequency optimization, tumor-bearing mice received subcutaneous injections of Rhodamine-6G (0.3 mL bolus) at concentrations of 10⁻⁶ M, 10⁻⁷ M, or 10⁻⁸ M either once a week or twice a week. [1]
For timing optimization, tumor-bearing mice received subcutaneous injections of 10⁻⁶ M Rhodamine-6G twice a week, starting either 7 days before, concurrently with, 7 days after, or 14 days after tumor cell implantation. [1]
The vehicle for Rhodamine-6G injections was PBS (pH 7.4), administered in a 0.3 mL bolus. Control animals received PBS injections without Rhodamine-6G. [1]
Tumor volume was assessed twice weekly using caliper measurements and calculated using the formula for a hemi-ellipsoid: V = π/6 × L × W × H, where L = length, W = width, and H = height. [1]
Animals were evaluated weekly for clinical status (appearance, attitude, appetite, thirst, urination, defecation), body weight, and tumor development. Survival was recorded, and at the endpoint, animals were sacrificed to assess abdominal metastases. [1]

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Three-month-old male C57Bl mice (25 ± 5 g) were used. B16-F10 melanoma cells (1.5 × 10⁶ cells in 0.2 mL PBS) were implanted subcutaneously. [1]
For dose-finding, tumor-bearing mice received subcutaneous injections of Rhodamine-6G (0.3 mL bolus) once a week at concentrations ranging from 10⁻¹⁰ M to 10⁻² M. Based on results, concentrations of 10⁻⁶ M, 10⁻⁷ M, and 10⁻⁸ M were selected for further study. [1]
For frequency optimization, tumor-bearing mice received subcutaneous injections of Rhodamine-6G (0.3 mL bolus) at concentrations of 10⁻⁶ M, 10⁻⁷ M, or 10⁻⁸ M either once a week or twice a week. [1]
For timing optimization, tumor-bearing mice received subcutaneous injections of 10⁻⁶ M Rhodamine-6G twice a week, starting either 7 days before, concurrently with, 7 days after, or 14 days after tumor cell implantation. [1]
The vehicle for Rhodamine-6G injections was PBS (pH 7.4), administered in a 0.3 mL bolus. Control animals received PBS injections without Rhodamine-6G. [1]
Tumor volume was assessed twice weekly using caliper measurements and calculated using the formula for a hemi-ellipsoid: V = π/6 × L × W × H, where L = length, W = width, and H = height. [1]
Animals were evaluated weekly for clinical status (appearance, attitude, appetite, thirst, urination, defecation), body weight, and tumor development. Survival was recorded, and at the endpoint, animals were sacrificed to assess abdominal metastases. [1]

Absorption, Distribution and Excretion
Rhodamine 6G exhibits high plasma protein binding. It has been reported that after intravenous infusion of 1 mg/min dye into dogs (strain, age, and sex unspecified), both Rhodamine 6G and Rhodamine B are excreted in situ via pancreatic juice, followed by secretin or cholecystokinin-pancreatic enzyme stimulation. However, the excretion rate has not been reported.

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In vitro, Rhodamine-6G at a concentration of 10⁻⁶ M was non-toxic to normal control cell cultures, with 90-95% of cells surviving after 5 days of treatment. [1]
In vivo, healthy (non-tumor-bearing) mice receiving subcutaneous injections of Rhodamine-6G at concentrations of 10⁻⁸ M, 10⁻⁷ M, and 10⁻⁶ M expressed no adverse responses to treatment and did not differ from untreated healthy controls. [1]
Healthy mice receiving higher concentrations of Rhodamine-6G (≥ 10⁻⁵ M) showed dose-dependent signs of adverse reactions. Animals receiving the highest doses (10⁻³ M and 10⁻² M twice a week) demonstrated toxic effects within the first 3 out of 5 weeks of the experiment. [1]

[1]. Low concentrations of Rhodamine 6G selectively destroy tumor cells and improve survival of melanoma transplanted mice. Neoplasma. 2013;60(3):262-73.

[2]. Fluorescence spectroscopy of Rhodamine 6G: concentration and solvent effects. Spectrochim Acta A Mol Biomol Spectrosc. 2014;121:147-51.

[3]. Low concentrations of Rhodamine 6G selectively destroy tumor cells and improve survival of melanoma transplanted mice. Neoplasma. 2013;60(3):262-73.

[4]. Measuring leukocyte-endothelial interactions in mice. Cold Spring Harb Protoc. 2013 Jun 1;2013(6):561-3.

Ci Basic Red 1 is a bright blue-pink crystal or a reddish-purple powder. (NTP, 1992)
Rhodamine 6G is rhodamine 6G(1+), an organochlorine salt and xatonium dye. It is used as a fluorescent dye.

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Rhodamine-6G is a fluorescent organic dye that binds to the inner membranes of mitochondria, reducing the number of intact, metabolically active mitochondria and inhibiting mitochondrial oxidative phosphorylation. This disrupts the respiratory chain, leading to cell death. [1]
The proposed mechanism for the selective anti-tumor activity of low-concentration Rhodamine-6G is based on the Warburg effect, where malignant cells have a reduced number of mitochondria per cell and an altered energy metabolism (relying more on glycolysis) compared to normal cells. Destroying a small number of mitochondria is hypothesized to be critical for energy-deprived tumor cells but not for normal cells with a higher mitochondrial reserve. [1]

C28H31CLN2O3

479.0103

478.202

989-38-8

13161-28-9 (perchlorate);63022-06-0 (molbdosilicate);63022-07-1 (molybdophosphate);63022-08-2 (tungstophosphate);65366-87-2 (molybdate)

13806

Light brown to red-brown solid powder

1.15g/cm3

<200ºC

290 °C

318.6ºC

1.593

7.225

2

5

7

34

823

0

VYXSBFYARXAAKO-UHFFFAOYSA-N

InChI=1S/C28H30N2O3.ClH/c1-6-29-23-15-25-21(13-17(23)4)27(19-11-9-10-12-20(19)28(31)32-8-3)22-14-18(5)24(30-7-2)16-26(22)33-25;/h9-16,29H,6-8H2,1-5H3;1H

[9-(2-ethoxycarbonylphenyl)-6-(ethylamino)-2,7-dimethylxanthen-3-ylidene]-ethylazanium;chloride

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ~25 mg/mL (~52.19 mM)
H2O : ~10 mg/mL (~20.88 mM)

Solubility in Formulation 1: 2.5 mg/mL (5.22 mM) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with sonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: 2.5 mg/mL (5.22 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: 4 mg/mL (8.35 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication (<60°C).

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 (Please use freshly prepared in vivo formulations for optimal results.)