Cationic fluorescent dye

TMRE Experimental Protocol
1. TMRE Solution Preparation
1.1 Stock Solution Preparation
Dissolve 1 mg TMRE in DMSO to prepare a 5 mM stock solution.

1.2 Working Solution Preparation
Dilute the stock solution with serum-free cell culture medium or PBS to obtain a working solution with a concentration range of 1–20 μM.
Note: Adjust the working solution concentration according to experimental requirements.
________________________________________
2. Cell Staining Procedure
2.1 Suspension Cell Treatment (6-well plate)
a. Cell Processing: Centrifuge at 1000g, 4°C for 3–5 min, discard supernatant. Wash twice with PBS, 5 min each. Adjust cell density to 1×10⁶/mL.
b. Staining: Add 1 mL working solution and incubate at room temperature for 5–30 min.
c. Centrifugation: Centrifuge at 400g, 4°C for 3–4 min, discard supernatant.
d. Washing: Wash twice with PBS, 5 min each.
e. Detection Preparation: Resuspend cells in serum-free medium or PBS for observation under a fluorescence microscope or flow cytometry.

2.2 Adherent Cell Treatment
a. Cell Culture: Culture adherent cells on sterile coverslips.
b. Pre-treatment: Remove coverslips and aspirate excess medium.
c. Staining: Add 100 μL working solution, gently shake to ensure complete coverage, and incubate at room temperature for 30–60 min.
d. Washing: Wash twice with culture medium, 5 min each.
e. Detection: Observe directly under a fluorescence microscope or analyze by flow cytometry.
Special Note: For flow cytometry analysis, cells must be resuspended before staining.

Confocal microscopy and 3D reconstruction [2]
To investigate mitochondrial movement, the mitochondria specific fluorescent dye tetramethylrhodamine ethyl ester (TMRE) was used. TMRE is a lipophilic, cell permeable, cationic, nontoxic, fluorescent dye that specifically stains live mitochondria. TMRE is accumulated specifically by the mitochondria in proportion to membrane potential. Whole lenses were loaded with TMRE by bathing the lens for 15 min at room temperature in 10 ml serum free M199 containing 5 μg/ml TMRE. Lenses were then mounted in 1% agarose on glass bottom plates as described previously. Time series and z-stacks of lens epithelial and superficial cortical fiber cells were acquired with a Zeiss 510 (configuration Meta 18) confocal laser scanning microscope equipped with an inverted Axiovert 200M microscope and 40x water immersion C-Apochromat objective (NA 1.2). The combination of an 488 nm Argon laser and 505 long pass emission filters were used to visualize TMRE fluorescence. Subsequent image restoration and analysis (such as the length of mitochondria and rate of movement) was done with commercial LSM510 VisArt and Physiology software packages (version 3.2; Carl Zeiss Inc., Jena, Germany). The representative length of mitochondria was measured from one end of the most visible structure to the other end using the measure function of software. The rate of movement (expressed in μm/sec) was determined by noticing the initial position (position zero) of moving mitochondria in the first X, Y gallery of time series, to the final movement observed during the time series by measuring the length in microns (μm).

Results [2]
The morphology and distribution of the mitochondria between lens epithelial and superficial cortical fiber cells is very different. In epithelial cells, up to 10 μm dense numerous perinuclear mitochondria, resembling an interconnected network were seen (Figure 1A). These dense mitochondria appear to fill the whole volume of the lens epithelial cell (Figure 2). In the superficial cortex the mitochondria were not as dense (Figure 1B), were more elongated (up to 65 μm), distinctly separated, and often branched (Figure 3).

Dynamic multidirectional movement of TMRE was observed in epithelial cells and bidirectional movement was seen in the superficial cortical fiber cells. In the epithelium, the movement of TMRE fluorescence was up to 5 μm/min (Figure 4) whereas in the superficial cortex the observed movement was up to 18.5 μm/min (Figure 5). The movement of TMRE fluorescence was abolished in both the lens epithelium (Figure 6) and the superficial cortex (Figure 7) following treatment with the uncoupler of the electron transport chain potential, CCCP.

---

Discussion [2]
The vertebrate lens is a cellular structure that consists of two types of cells organized in distinct spatial patterns; the epithelial monolayer that covers the anterior surface and fiber cells that comprise the bulk of the lens. Cell division in the lens is restricted to the epithelial cells. This monolayer can be divided into three compartments, related to lens anatomy and rate of cell division. Central epithelial cells overlying the anterior suture regions of the lens normally undergo little or no mitosis. Intermediately located epithelial cells undergo limited mitosis whereas epithelial cells at the lens equator show the fastest mitotic rate. These equatorial epithelial cells give rise to terminally differentiated fiber cells in a process that continues throughout life, resulting in a steady increase in tissue volume.

Highly variable morphology and distribution of mitochondria is observed in different types of cells. In growing cells mitochondria are frequently found as extremely dynamic structures with tubular sections dividing in half, branching, and fusing to form a complex network. In differentiated cells, such as cardiac muscle or kidney tubules, mitochondria are often localized in specific cytoplasmic regions rather than randomly distributed. In this study, three dimensional reconstruction of the lens epithelium indicates that mitochondria may form a complex network (Figure 2) similar to that of other mammalian cells. In contrast to the lens epithelium the mitochondria of the superficial cortical fiber cells were not as dense, were distinctly separated and often branched (Figure 3 and Figure 5). Despite such variability between the two cell types, no obvious difference in morphology or distribution is observed between lenses of different species, including fish and mammals. The random distribution of the mitochondria throughout the terminally differentiated superficial cortical fiber cells (Figure 1B, Figure 3, and Figure 5) also contrasts with the more localized distribution seen in other differentiated cells such as those of cardiac muscle or kidney tubules.

Using confocal laser scanning microscopy of excised bovine lenses stained with the mitochondria specific dye tetramethylrhodamine ethyl ester (TMRE), this study is the first to show the dynamic movement of TMRE in both lens epithelial cells and in the superficial cortex.

General observations of mitochondria go back as far as the middle of the nineteenth century. The observed structures were often called granules and in 1898 a German microbiologist Carl Benda coined the name "mitochondrion" from Greek mitos "thread" and khondrion "little granule". In living cells the mitochondria are dynamic structures continually moving and changing their size and shape. One of the earlier observations of the mitochondrial dynamic reported in the literature goes back to 1915 when Lewis and Lewis using cultured embryonic chick cells and light microscopy reported movement of the mitochondria in living cells. These observations supported suggestions that mitochondria were related to bacteria, foreshadowing widespread acceptance of the endosymbiotic theory of mitochondrial descent from prokaryotic cells that were symbiotically established in the cytoplasm of eukaryotic cells. Early clues to the mechanisms of mitochondrial distribution and movement emerged from studies of the cytoskeleton. Microscopic analysis revealed colocalization of mitochondria with certain cytoskeletal components. In particular, many studies documented colocalization of mitochondria with microtubules in diverse cell types including mammalian neurons, cultured fibroblasts, and the protozoan Acanthamoeba castellanii. Involvement of microtubules was further supported by the observation that mitochondria redistribute in cultured mammalian cells treated with agents that disassemble microtubule networks. Furthermore, disruption of microtubules by certain conditional mutations in genes encoding tubulins (the building blocks of microtubules) caused aberrant mitochondrial distribution in S. pombe, providing genetic evidence suggesting that microtubules position mitochondria in this organism. A pivotal advance in identifying the molecular basis of organelle movement on microtubules was the discovery of the microtubule based motor proteins, kinesin and cytoplasmic dynein. These proteins appear to bind microtubules and transduce chemical energy (ATP) into mechanical work to power polarized movement of the mitochondria along microtubules. Both proteins can bind and transport "cargo" in the form of vesicles, organelles, or other proteins, and mitochondria appear to be among the favored cargoes. In particular, several different members of the kinesin superfamily have been localized preferentially to mitochondria in animal cells. A recent study of rat lens showed the existence of a microtubule based motor system containing both kinesin and dynein in the elongating fiber cells. Presence of this system was attributed to the transportation of important membrane proteins and organelles to the target regions during increased cell growth that accompanies elongation of the secondary fiber cells. A large number of microtubules were regularly arranged into bundles parallel to the long axis of fiber cells, a morphological observation similar to that of the distribution of the mitochondria seen in the superficial cortical fiber cells (Figure 1B) and may represent the machinery responsible for the observed rapid organelle movement.

The observed dynamics of TMRE fluorescence movement may represent actual mitochondrial movement, indicating the dynamic state of the mitochondria in both lens epithelium and superficial cortex. That this activity is found not only in the epithelium but also in the superficial cortex indicates that the superficial cortical fiber cells play a much more active role in lens metabolism than previously suspected. Alternatively, the observed movement of TMRE across a mitochondrial network could represent change in the distribution of potential across the inner membrane, presumably allowing energy transmission across the cell from regions of low to regions of high ATP demand.

In summary, this report describes a new observation regarding the dynamic properties of the mitochondria of the lens. Further research will probe the relation between mitochondrial dynamics and the microtubule based motor system. Furthermore, the effects of CCCP on mitochondrial dynamics suggest that this approach may be useful in evaluating lens integrity.

Eye dissection and treatment [2]
Bovine eyes, obtained from a local abattoir, were dissected, and the lenses were excised within 1-5 h post-mortem under sterile conditions, as described previously. Briefly, the lenses were placed in a three part chamber made from glass, silicon rubber, and a metal base and immersed in 21 ml of culture medium consisting of Medium 199 with Earle's salts, 100 mg/l L-glutamine, 3% dyalized fetal bovine serum, 2.2 g/l sodium bicarbonate, 5.96 g/l HEPES, and 1% antibiotics (100 units/ml penicillin and 0.1 mg/ml streptomycin). The cultured lenses were incubated at 37 °C and 4% CO2 for 48 h prior to experimental use and lenses that were damaged during dissection were excluded from the study.
To demonstrate the fluorescence specificity of TMRE, the uncoupler of the electron transport chain potential, carbonyl cyanide m-chlorophenylhydrazone (CCCP), was used. Lenses were transferred for treatment into glass vials containing 32.5 μM CCCP in 10 ml serum free M199 and incubated for 30 min. The lenses were then rinsed three times with physiological saline and culture medium.

[1]. Measuring Mitochondrial Transmembrane Potential by TMRE Staining. Cold Spring Harb Protoc. 2016 Dec 1;2016(12):pdb.prot087361.

[2]. Confocal laser scanning microscopy imaging of dynamic TMRE movement in the mitochondria of epithelial and superficial cortical fiber cells of bovine lenses. Mol Vis. 2005 Jul 14;11:518-23.

Tetramethylrhodamine ethyl ester perchlorate is an organic perchlorate salt that has tetramethylrhodamine ethyl ester(1+) as the cation. It is used as a cell-permeant, cationic, red-orange fluorescent dye that is readily sequestered by active mitochondria. It has a role as a fluorochrome and a reagent. It is a xanthene dye and an organic perchlorate salt. It contains a tetramethylrhodamine ethyl ester(1+).

---

Adenosine triphosphate (ATP) is the main source of energy for metabolism. Mitochondria provide the majority of this ATP by a process known as oxidative phosphorylation. This process involves active transfer of positively charged protons across the mitochondrial inner membrane resulting in a net internal negative charge, known as the mitochondrial transmembrane potential (ΔΨm). The proton gradient is then used by ATP synthase to produce ATP by fusing adenosine diphosphate and free phosphate. The net negative charge across a healthy mitochondrion is maintained at approximately -180 mV, which can be detected by staining cells with positively charged dyes such as tetramethylrhodamine ethyl ester (TMRE). TMRE emits a red fluorescence that can be detected by flow cytometry or fluorescence microscopy and the level of TMRE fluorescence in stained cells can be used to determine whether mitochondria in a cell have high or low ΔΨm. Cytochrome c is essential for producing ΔΨm because it promotes the pumping the protons into the mitochondrial intermembrane space as it shuttles electrons from Complex III to Complex IV along the electron transport chain. Cytochrome c is released from the mitochondrial intermembrane space into the cytosol during apoptosis. This impairs its ability to shuttle electrons between Complex III and Complex IV and results in rapid dissipation of ΔΨm. Loss of ΔΨm is therefore closely associated with cytochrome c release during apoptosis and is often used as a surrogate marker for cytochrome c release in cells.[1]

---

Purpose: Recent confocal laser scanning microscopy studies of the mitochondria of vertebrate lenses show a striking difference in the distribution and morphology of the mitochondria of lens epithelial and superficial cortical cells. This study, using confocal microscopy, was undertaken to image the movement of the mitochondria specific dye tetramethylrhodamine ethyl ester (TMRE) in the epithelium and superficial cortex of whole live bovine lens.
Methods: Cultured bovine lenses were loaded with 5 microg/ml TMRE for 15 min at room temperature. TMRE fluorescence was acquired with a Zeiss 510 (configuration META 18) confocal laser scanning microscope for 10 to 15 min using 488 nm Argon laser excitation and 505 nm long pass emission filter settings. The uncoupler of the electron transport chain potential, carbonyl cyanide m-chlorophenylhydrazone (CCCP, 32.5 microM), was used to demonstrate the fluorescent specificity of TMRE.
Results: Multidirectional dynamic movement of TMRE was observed in epithelial cells and bidirectional dynamic movement was seen in the superficial cortical fiber cells of live bovine lenses. In the epithelium, the movement of TMRE fluorescence was up to 5 microm/min whereas in the superficial cortex the observed movement was up to 18.5 microm/min. The movement of TMRE fluorescence was abolished with treatment with the uncoupler, CCCP.
Conclusions: The observed dynamics of TMRE fluorescence movement may represent actual mitochondrial movement, indicating the dynamic state of the mitochondria in both lens epithelium and superficial cortex. That this activity is found not only in the epithelium but also in the superficial cortex indicates that the superficial cortical fiber cells play a much more active role in lens metabolism than previously suspected. Alternatively, the observed movement of TMRE across a mitochondrial network could represent change in the distribution of potential across the inner membrane, presumably allowing energy transmission across the cell from regions of low to regions of high ATP demand [2].

C26H27CLN2O7

514.9548

514.15

115532-52-0

2762682

Brown to black solid powder

6.057

0

8

5

36

884

0

NBAOBNBFGNQAEJ-UHFFFAOYSA-M

InChI=1S/C26H27N2O3.ClHO4/c1-6-30-26(29)20-10-8-7-9-19(20)25-21-13-11-17(27(2)3)15-23(21)31-24-16-18(28(4)5)12-14-22(24)25;2-1(3,4)5/h7-16H,6H2,1-5H3;(H,2,3,4,5)/q+1;/p-1

[6-(dimethylamino)-9-(2-ethoxycarbonylphenyl)xanthen-3-ylidene]-dimethylazanium;perchlorate

115532-52-0; Tetramethylrhodamine ethyl ester perchlorate; tetramethylrhodamine ethyl ester; TMRE perchlorate; Xanthylium, 3,6-bis(dimethylamino)-9-[2-(ethoxycarbonyl)phenyl]-, perchlorate (1:1); CHEBI:78720; DTXSID70376365; Xanthylium, 3,6-bis(dimethylamino)-9-(2-(ethoxycarbonyl)phenyl)-, perchlorate (1:1); DTXCID30327393;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture and light.

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

DMSO : ~27.78 mg/mL (~53.95 mM)

Solubility in Formulation 1: ≥ 2.08 mg/mL (4.04 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 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.

---

Solubility in Formulation 2: ≥ 2.08 mg/mL (4.04 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 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.

---

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