TMRM Perchlorate

• CAT Number: M022874
• CAS Number: 115532-50-8
• Molecular Formula: C25H25ClN2O7
• Molecular Weight: 500.93
• Purity: ≥95%
• Synonyms: [6-(dimethylamino)-9-(2-methoxycarbonylphenyl)xanthen-3-ylidene]-dimethylazanium;perchlorate
• InChI: InChI=1S/C25H25N2O3.ClHO4/c1-26(2)16-10-12-20-22(14-16)30-23-15-17(27(3)4)11-13-21(23)24(20)18-8-6-7-9-19(18)25(28)29-5;2-1(3,4)5/h6-15H,1-5H3;(H,2,3,4,5)/q+1;/p-1
• InChIKey: PFYWPQMAWCYNGW-UHFFFAOYSA-M
• SMILES: CN(C)C1=CC2=C(C=C1)C(=C3C=CC(=[N+](C)C)C=C3O2)C4=CC=CC=C4C(=O)OC.[O-]Cl(=O)(=O)=O

Rhodamine dyes are membrane-permeable cationic fluorescent probes that specifically recognize mitochondrial membrane potentials, thereby attaching to mitochondria and producing bright fluorescence, and at certain concentrations, rhodamine dyes have low toxicity to cells, so they are commonly used to detect mitochondria in animal cells, plant cells, and microorganisms[1].
1.Preparation of TMRM working solution
1.1Preparation of the stock solution
Dissolve 1 mg TMRM in 525 μL DMSO to obtain 5 mM of stock solution.
1.2Preparation of TMRM working solution
Dilute the stock solution in serum-free cell culture medium or PBS to obtain 1-20 μM of working solution.
Note: Please adjust the concentration of TMRM working solution according to the actual situation.
2.Cell staining
2.1 Suspension cells (6-well plate)
a.Centrifuge at 1000 g at 4℃ for 3-5 minutes and then discard the supernatant. Wash twice with PBS, 5 minutes each time.The cell density is 1×106/mL.
b.Add 1 mL of working solution, and then incubate at room temperature for 5-30 minutes.
c.Centrifuge at 400 g at 4℃ for 3-4 minutes and then discard the supernatant.
d.Wash twice with PBS, 5 minutes each time.
e.Resuspend cells with serum-free cell culture medium or PBS. Observation by fluorescence microscopy or flow cytometry.
2.2 Adherent cells
a.Culture adherent cells on sterile coverslips.
b.Remove the coverslip from the medium and aspirate excess medium.
c.Add 100 μL of working solution, gently shake it to completely cover the cells,and then incubate at room temperature for 30-60 minutes.
d.Wash twice with medium, 5 minutes each time. Observation by fluorescence microscopy or flow cytometry.
Note: If detection by flow cytometry, cells need to be resuspended before staining.

Reference

[1]. Crowley LC, et al. Measuring Mitochondrial Transmembrane Potential by TMRE Staining. Cold Spring Harb Protoc. 2016 Dec 1;2016(12):pdb.prot087361.
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[2]. Chowdhury SR, et al. Simultaneous evaluation of substrate-dependent oxygen consumption rates and mitochondrial membrane potential by TMRM and safranin in cortical mitochondria. Biosci Rep. 2015 Dec 8;36(1):e00286.
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[3]. Monteith A, et al. Imaging of mitochondrial and non-mitochondrial responses in cultured rat hippocampal neurons exposed to micromolar concentrations of TMRM. PLoS One. 2013;8(3):e58059.
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