MAR

[Hypoxia detecting probe]

495-540 nm：Green

– For the study of cancer or ischemia –

Easily available for live-cell imaging
As sensitive as pimonidazole
Available for flow cytometry

Hypoxia in living tissue closely relates to a wide array of pathologies, including cancer and ischemia. When using with the existing hypoxia detection probe, pimonidazole, immunostaining is necessary, and the probe is only available for the fixed cells. However, live-cell imaging of hypoxia is easily available with MAR.

Available through Merck KGaA (Darmstadt, Germany) as:
SCT033 BioTracker™ 520 Green Hypoxia Dye

Products

Code No.	Product Name	Size	Merck CAT No.	Merck ( Millipore / Sigma Aldrich ) Product Name
A101-01	MAR	25 μg × 5	SCT033	BioTracker 520 Green Hypoxia Dye

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MAR
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Properties of MAR

Product Name
target Reactivity Cell permeability
Abs_max (nm) FL_max (nm)

MAR hypoxia irreversible Yes 498 520

Compatibility for live-cell imaging

Fluorescent intensity increased as content of oxygen decreased. Pimonidazole is used to be generally used for the detection of hypoxia, but cell fixation and immunostaining are necessary. However, fluorescent live-cell imaging of hypoxia is available only by addition of MAR to the living cells.

Fig. 1. Reduced reaction of MAR under hypoxia

Though MAR is non-fluorescent, reductive cleavage in the azo base of the probe occurs by the reductive enzyme under hypoxia, and 2Me RG is generated which makes bright green fluorescence. 2Me RG tends to localize in mitochondria because of its positive charge.

As sensitive as pimonidazole

Fig. 2. Fluorescent intensity in A549 cell under hypoxia, stained by MAR or pimonidazole.

A549 cells were observed after stained by MAR or pimonidazole under the various concentrations of oxygen. While pimonidazole responded to the concentration of oxygen under the 1%, MAR responded to the oxygen concentration of about 5%.
Hypoxia imaging in spheroid using FerroOrange and MAR
MAR
Hypoxia imaging in spheroid using FerroOrange and MAR

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Hypoxia imaging in spheroid using FerroOrange and MAR

It has been reported that in a decreased oxygen partial pressure condition (hypoxia), intracellular labile Fe²⁺ increases compared to that in a normoxia conditions (T. Hirayama et al., 2017, Chem. Sci. 8: 4858-4866). Here we observed hypoxia and intracellular labile Fe²⁺ increase using fluorescent probes, FerroOrange and MAR, simultaneously.

To spheroids of HepG2 cells, MAR (final 1 μM) was applied and cultured for 4 hours at 37℃, 5% CO₂. Furthermore, FerroOrange (final 1 µM) was also applied and cultured for 30 minutes. Strong fluorescence derived from FerroOrange was observed in the center region of spheroids (left, red). Fluorescence of MAR (center, green) was also detected in the central regions. Overlayed image (right) indicates that the both area are almost overlapped, indicating that cellular hypoxic response and increase in labile Fe²⁺ occurs in the almost same regions.

Experiment procedure
1. Spheroid of HepG2 cells were formed using cell culture dishes to which cells do not adhere. Alternatively, commercially available spheroid-forming kits can be also used. Refer the manufacture’s instructions for the procedure.
2. Carefully transfered the spheloids to glass-bottomed dishes, and cultured for ~1 days to let the spheloids adhere to the dish.
3. Added MAR to the medium (final 1 μM) and cultured for 4 hours at 37℃, 5% CO₂.
4. Added FerroOrange to the medium (final 1 μM) and cultured for 30 minutes at 37℃, 5% CO₂.
5. Carefully rinsed spheloids with HBSS twice, not to removing spheloids from the bottom, and observed by fluorescence microscopy.

FAQ

Q How many assays are available for 25 μg of MAR?

A

From a vial of MAR (25 μg) , ~43 μL of 1 mM stock solution can be made. If you use 1 mL of 1 μM MAR solution for one assay, you can perform ~40 assays per one vial.
Q Which reductase is responsible for the reaction of MAR?

A

It is not precisely determined, however, NADPH cytochrome P450 is one of the candidate because diphenyliodonium chloride has been reported to inhibit the reaction of MAR.
Q Can I measure the oxygen concentration using MAR?

A

Unfortunately, no. Nonfluorescent MAR is digested by intracellular reductase which is activated by low oxygen concentration to form fluorescent substance. Based on the complicated reaction mechanism, it is not easy to prepare calibration curve to determine the oxygen concentration.
Q Does oxygen-concentration increase during flow cytometric analysis change the fluorescence of MAR?

A

MAR, a material with no fluorescence fluoresces in hypoxic environment. Because this reaction is irreversible, you can dissociate cells by trypsin and dilute cells into normal buffers or medium without decreasing the MAR’s fluorescence.
Q Can I observe MAR fluorescence after fixation?

A

Yes, mild fixation does not bleach the fluorescence, however, strong fixation condition may alter the intracellular localization of MAR or decrease the fluorescence. Please test the fixation condition in your cells and conditions.