Ca2+ chelator; Chelating Agents

Guidelines[1] (Listed below is our recommended protocol, which provides a guideline and may be adjusted based on your specific needs).
Labeling of Cells:
1. Incubate the cells using standard protocols.
2. Pre-incubate cells for 30 min with 20 μM MAPTAM.
3. Replace medium with HEPES buffer containing 2 μM fura 2 pentaacetoxymethyl ester and 1% BSA (wt:vol).
4. Conduct measurements in HEPES buffer containing 1 mM CaCl2.

[1]. Role of mitogen-activated protein kinase in the angiotensin II-induced DNA synthesis in vascular smooth muscle cells. Hypertension. 1998 May;31(5):1151-6.

Chelating agents are chemical substances that bind to and remove ions in solution. Many chelating agents function by forming coordination complexes with metals. The activation of mitogen-activated protein kinase (MAP) and the increase in intracellular free calcium ion concentration ([Ca2+]i) are associated with the activation of various protein kinases and the growth of vascular smooth muscle cells (VSMCs). This study aimed to investigate the role of angiotensin II-induced increases in [Ca2+]i in the activation of 44-kD/42-kD MAP kinases (p44mapk/p42mapk) and DNA synthesis in VSMCs. The experiments used the intracellular chelating agent 1,2-bis-(o-amino-5-methylphenoxy)ethane-N,N,N',N'-tetraacetate tetraacetoxymethyl ester (MAPTAM) to chelate [Ca2+]i. Ca2+ concentration was determined using the Fura-2 assay. MAP kinase activation was detected using Western blotting. DNA synthesis was determined by measuring the amount of [3H]thymidine incorporated into cellular DNA. After VSMCs were treated with 20 μmol/L MAPTAM for 30 minutes, the maximum increase induced by Ang II at 10 seconds completely disappeared. Ang II phosphorylates p44MAPK/p42MAPK in a time-dependent manner, peaking at 3 minutes. In MAPTAM-treated cells, the maximum phosphorylation time point of the MAP kinase isoform shifted to 5 minutes, and dephosphorylation was delayed compared to untreated cells. Consistent with this result, the induction of MAP kinase phosphatase-1 was significantly impaired in MAPTAM-treated cells. In untreated cells, Ang II increased the amount of [3H]thymidine incorporated into DNA synthesis by 2.3-fold. This effect was not attenuated in MAPTAM-treated cells. Treatment of cells with the MAP kinase inhibitor PD 98059 (10 μmol/L) inhibited Ang II-induced MAP kinase activation by up to 85%, but did not inhibit Ang II-induced DNA synthesis. In summary, Ang II-induced MAP kinase activation is a Ca2+-dependent process. Furthermore, blocking the activation of early intracellular events induced by Ang II (such as [Ca2+]i elevation or MAP kinase phosphorylation) is not accompanied by inhibition of Ang II-induced DNA synthesis. [1]

C36H44N2O18

792.74

792.259

147504-94-7

3536452

Typically exists as solid at room temperature

1.629

0

20

31

56

1160

0

CC(OCOC(CN(C1=CC=C(C)C=C1OCCOC1=CC(C)=CC=C1N(CC(OCOC(=O)C)=O)CC(OCOC(=O)C)=O)CC(OCOC(=O)C)=O)=O)=O

HEOJVQZWOLQUDR-UHFFFAOYSA-N

InChI=1S/C36H44N2O18/c1-23-7-9-29(37(15-33(43)53-19-49-25(3)39)16-34(44)54-20-50-26(4)40)31(13-23)47-11-12-48-32-14-24(2)8-10-30(32)38(17-35(45)55-21-51-27(5)41)18-36(46)56-22-52-28(6)42/h7-10,13-14H,11-12,15-22H2,1-6H3

acetyloxymethyl 2-[N-[2-(acetyloxymethoxy)-2-oxoethyl]-2-[2-[2-[bis[2-(acetyloxymethoxy)-2-oxoethyl]amino]-5-methylphenoxy]ethoxy]-4-methylanilino]acetate

maptam; 147504-94-7; 5 5/'-DIMETHYL-BAPTA-AM*; acetyloxymethyl 2-[N-[2-(acetyloxymethoxy)-2-oxoethyl]-2-[2-[2-[bis[2-(acetyloxymethoxy)-2-oxoethyl]amino]-5-methylphenoxy]ethoxy]-4-methylanilino]acetate; Tetrakis(acetoxymethyl) 2,2',2'',2'''-(((ethane-1,2-diylbis(oxy))bis(4-methyl-2,1-phenylene))bis(azanetriyl))tetraacetate; SCHEMBL13235775; MFCD00036856;

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)

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

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.

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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).

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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)

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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).

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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

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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.

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