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Pimonidazole is a novel hypoxia marker for complementary study of tumor hypoxia and cell proliferation in tumor. Pimonidazole accumulates in hypoxic cells via covalent binding with macromolecules or by forming reductive metabolites after reduction of its nitro group, it can be used for qualitative and quantitative assessment of tumor hypoxia .

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Pimonidazole hydrochloride Chemical Structure

Pimonidazole hydrochloride Chemical Structure

CAS No. : 70132-51-3

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10 mM * 1 mL in DMSO
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Based on 4 publication(s) in Google Scholar

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Description

Pimonidazole is a novel hypoxia marker for complementary study of tumor hypoxia and cell proliferation in tumor[1]. Pimonidazole accumulates in hypoxic cells via covalent binding with macromolecules or by forming reductive metabolites after reduction of its nitro group, it can be used for qualitative and quantitative assessment of tumor hypoxia [2].

In Vitro

Pimonidazole, the exogenous hypoxia marker, is a 2-nitroimidazole compound, which forms covalent bonds with cellular macromolecules at oxygen levels below 1.3%.
Detection: Hypoxic cells were recognized by immunohistochemical detection of pimonidazole using a mouse monoclonal antibody. Cell proliferation was detected with a commercially available monoclonal antibody for proliferating cell nuclear antigen (PCNA). Assessment of hypoxia and cell proliferation was made qualitatively with light microscopy and quantitatively using point counting and image analysis software methods.
The pimonidazole-protein adducts were then detected by immunofluorescence
Cells (90% confluent) are treated with pimonidazole hydrochloride (10-100 μM), diluted in complete media or PBS, and placed in hypoxia or normoxia for 2-4 hours.
Then, cells are washed 4 times in HBSS, fixed for 10 min with 10% neutral buffered formalin at room temperature, washed 3 times with tris buffered saline, and mounted with Prolong Gold with DAPI mounting media[3].
To monitor the intrinsic hypoxic signal, the tissue is removed and the embryos incubated with 400 μM pimonidazole for 2 h before fixation. Immunostaining is performed using antibodies against pimonidazole-protein adducts (Hypoxyprobe)[4].

MedChemExpress (MCE) has not independently confirmed the accuracy of these methods. They are for reference only.

In Vivo

Pimonidazole is a 2-nitroimidazole that is reductively activated specifically in hypoxic cells and forms stable adducts with thiol groups in proteins, peptides, and amino acids. Additionally, the amount of pimonidazole that is detected is directly proportional to the level of hypoxia within tumors in vivo.
The following protocol is provided for this useful tool to interrogate the levels of hypoxia in vivo.
1.Pimonidazole hydrochloride is suspended at a concentration of 30 mg/ml in 0.9% sterile saline or PBS.
2.Multiple mice are injected intravenously (tail vein) with 60 mg/kg of the pimonidazole solution.
3.Let circulate in vivo for 90 min before the mice are euthanazied with isoflurane or carbon dioxide (CO2). Note: For animal euthanasia: In compliance with proper IACUC protocols!
4. Mice are sacrificed and organs are then removed, weighed, snap-frozen, and stored in plastic scintillation vials at -80 °C.
5. The pimonidazole stain can work on paraffin-embedded tissue, frozen tissue, and cell lines. Organs are embedded in OCT, placed in a cryostat, and cut into 10 μm-thick sections. Sections are placed on microscope slides for staining.

MedChemExpress (MCE) has not independently confirmed the accuracy of these methods. They are for reference only.

Clinical Trial
Molecular Weight

290.75

Formula

C11H19ClN4O3

CAS No.
Appearance

Solid

Color

White to yellow

SMILES

OC(CN1C=CN=C1[N+]([O-])=O)CN2CCCCC2.Cl

Shipping

Room temperature in continental US; may vary elsewhere.

Storage

4°C, sealed storage, away from moisture

*In solvent : -80°C, 6 months; -20°C, 1 month (sealed storage, away from moisture)

Solvent & Solubility
In Vitro: 

DMSO : 250 mg/mL (859.85 mM; Need ultrasonic; Hygroscopic DMSO has a significant impact on the solubility of product, please use newly opened DMSO)

H2O : 5 mg/mL (17.20 mM; ultrasonic and warming and heat to 80°C)

Preparing
Stock Solutions
Concentration Solvent Mass 1 mg 5 mg 10 mg
1 mM 3.4394 mL 17.1969 mL 34.3938 mL
5 mM 0.6879 mL 3.4394 mL 6.8788 mL
View the Complete Stock Solution Preparation Table

* Please refer to the solubility information to select the appropriate solvent. Once prepared, please aliquot and store the solution to prevent product inactivation from repeated freeze-thaw cycles.
Storage method and period of stock solution: -80°C, 6 months; -20°C, 1 month (sealed storage, away from moisture). When stored at -80°C, please use it within 6 months. When stored at -20°C, please use it within 1 month.

* Note: If you choose water as the stock solution, please dilute it to the working solution, then filter and sterilize it with a 0.22 μm filter before use.

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  • Dilution Calculator

Mass (g) = Concentration (mol/L) × Volume (L) × Molecular Weight (g/mol)

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Concentration (start) × Volume (start) = Concentration (final) × Volume (final)

This equation is commonly abbreviated as: C1V1 = C2V2

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In Vivo:

For the following dissolution methods, please prepare the working solution directly. It is recommended to prepare fresh solutions and use them promptly within a short period of time.
The percentages shown for the solvents indicate their volumetric ratio in the final prepared solution. If precipitation or phase separation occurs during preparation, heat and/or sonication can be used to aid dissolution.

  • Protocol 1

    Add each solvent one by one:  50% PEG300    50% Saline

    Solubility: 10 mg/mL (34.39 mM); Clear solution; Need ultrasonic

In Vivo Dissolution Calculator
Please enter the basic information of animal experiments:

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Number of animals

Recommended: Prepare an additional quantity of animals to account for potential losses during experiments.
Calculation results:
Working solution concentration: mg/mL
Purity & Documentation

Purity: 99.96%

References

Complete Stock Solution Preparation Table

* Please refer to the solubility information to select the appropriate solvent. Once prepared, please aliquot and store the solution to prevent product inactivation from repeated freeze-thaw cycles.
Storage method and period of stock solution: -80°C, 6 months; -20°C, 1 month (sealed storage, away from moisture). When stored at -80°C, please use it within 6 months. When stored at -20°C, please use it within 1 month.

Optional Solvent Concentration Solvent Mass 1 mg 5 mg 10 mg 25 mg
H2O / DMSO 1 mM 3.4394 mL 17.1969 mL 34.3938 mL 85.9845 mL
5 mM 0.6879 mL 3.4394 mL 6.8788 mL 17.1969 mL
10 mM 0.3439 mL 1.7197 mL 3.4394 mL 8.5985 mL
15 mM 0.2293 mL 1.1465 mL 2.2929 mL 5.7323 mL
DMSO 20 mM 0.1720 mL 0.8598 mL 1.7197 mL 4.2992 mL
25 mM 0.1376 mL 0.6879 mL 1.3758 mL 3.4394 mL
30 mM 0.1146 mL 0.5732 mL 1.1465 mL 2.8662 mL
40 mM 0.0860 mL 0.4299 mL 0.8598 mL 2.1496 mL
50 mM 0.0688 mL 0.3439 mL 0.6879 mL 1.7197 mL
60 mM 0.0573 mL 0.2866 mL 0.5732 mL 1.4331 mL
80 mM 0.0430 mL 0.2150 mL 0.4299 mL 1.0748 mL
100 mM 0.0344 mL 0.1720 mL 0.3439 mL 0.8598 mL

* Note: If you choose water as the stock solution, please dilute it to the working solution, then filter and sterilize it with a 0.22 μm filter before use.

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  • Do most proteins show cross-species activity?

    Species cross-reactivity must be investigated individually for each product. Many human cytokines will produce a nice response in mouse cell lines, and many mouse proteins will show activity on human cells. Other proteins may have a lower specific activity when used in the opposite species.

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Pimonidazole hydrochloride
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