KMG-301AM TFA is the acetoxymethyl esterified form of KMG-301. KMG-301AM TFA successfully accumulates in mitochondria and then hydrolyzes to KMG-301. KMG-301 is a Mg2+ selective fluorescent probe that functions in mitochondria of intact cells. Since KMG-301 cannot permeate the mitochondrial membrane, KMG-301 is not released when the mitochondrial membrane potential is depolarized. KMG-301 can indicate mitochondrial Mg2+ concentration and can show Mg2+ transport across the mitochondrial membrane in the early stages of cell models.

Physicochemical Properties

Molecular Formula	C32H28F3N3O8
Molecular Weight	639.58
CAS #	1372642-78-8
PubChem CID	171038163
Appearance	Solid powder
Hydrogen Bond Acceptor Count	12
Rotatable Bond Count	7
Heavy Atom Count	46
Complexity	1400
Defined Atom Stereocenter Count	0
HS Tariff Code	2934.99.9001
Storage	Powder-20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month Note: (1). This product requires protection from light (avoid light exposure) during transportation and storage.(2). Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture.
Shipping Condition	Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Biological Activity

ln Vitro

Protocol: This protocol is intended only as a guide and should be modified to suit your specific needs [1]. 1. For optical imaging, incubate cells with 20 µM KMG-301AM TFA in Hanks balanced salt solution (HBSS) on ice for 10 min to avoid hydrolysis of acetoxymethyl esters by esterases present in the cytoplasm. HBSS contains (mM): NaCl, 137: KCl, 5.4; CaCl2, 1.3; MgCl2, 0.5; MgSO4, 0.4; Na2HPO4, 0.3; KH2PO4, 0.4; NaHCO3, 4.2; D-glucose, 5.6; HEPES, 5 (pH adjusted to 7.4 with NaOH). 2. Wash cells twice with HBSS and incubate at 37°C for 15 min to allow complete hydrolysis of acetoxymethyl ester forms in mitochondria. 3. If you want to use KMG-104AM (HY-128536) and KMG-301AM TFA at the same time, incubate the cells with 5 µM KMG-104AM in HBSS at 37°C for 30 minutes, and then stain with KMG-301AM TFA. 4. Incubate the isolated mitochondria with 20 µM KMG-301AM TFA (dissolved in Mitochondrial Imaging Buffer (MIB)) at 37°C for 20 minutes. Then wash the mitochondria twice with MIB and further incubate at 37°C for 15 minutes. MIB contains (mM): KCl, 125; K2HPO4, 2; MgCl2, 1; HEPES, 5; EDTA, 0.02 (pH adjusted to 7.2 with KOH) or MIB without Mg2+ (without MgCl2). 5. Perform fluorescence imaging experiments using a confocal laser scanning microscope system. KMG-301 was excited by a laser diode at 559 nm and the signal was observed at 600-700 nm. In double staining, KMG-104 and KMG-301 were excited by an argon laser at 488 nm and 559 nm, respectively. The fluorescence was separated using a 560 nm dichroic mirror and observed at 500-545 nm and 600-700 nm, respectively.

References

[1]. Newly developed Mg2+-selective fluorescent probe enables visualization of Mg2+ dynamics in mitochondria. PLoS One. 2011;6(8):e23684.

Solubility Data

Solubility (In Vitro)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

Solubility (In Vivo)

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples. Injection Formulations (e.g. IP/IV/IM/SC) Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline) *Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution. Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline) Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil) Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals). Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)] *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. Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline) Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline) Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline) Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil) Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline) Oral Formulations Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium) Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals). Oral Formulation 3: Dissolved in PEG400 Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose Oral Formulation 6: Mixing with food powders Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.  (Please use freshly prepared in vivo formulations for optimal results.)

Preparing Stock Solutions		1 mg	5 mg	10 mg
	1 mM	1.5635 mL	7.8176 mL	15.6353 mL
	5 mM	0.3127 mL	1.5635 mL	3.1271 mL
	10 mM	0.1564 mL	0.7818 mL	1.5635 mL

*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.