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Hypoxia Probe

It is known that spheroids formed by cancer cell lines such as HT-29 exhibits hypoxic region inside the spheroid. Reporter gene assay, such as by integrating GFP gene to the downstream of hypoxia-responsible element, or using pimonidazole immune-staining adducts in hypoxic region is generally used for intracellular hypoxic-sensing. However, it requires to establish the cell lines with stable expression, and troublesome procedure for staining or section preparation.

Hypoxia Probe LOX-1 enables homogeneous assay for intracellular hypoxic-sensing, just by adding the reagent into the medium for 12 hr before measuring, and does not require excess reagent washing step. Hypoxic region inside the spheroid can be detected by observation with conventional fluorescence microscopes.


We also supply DMSO solution of Hypoxia Probe (2mM). Customer can add this reagent into culture medium at 1,000 times dilution.

Product Name Cat.# Qty Storage Document
Hypoxia Probe
LOX-1 2mg Room temp.,
Protection from light
Handbook

MSDS
Hypoxia Probe Solution LOX-1S 2 mM,
100 μL
-20℃,
Protection from light
Handbook

2D: Intracellular hypoxia sensing (comparison with HIF-1a expression level)

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Similar result with the immunostaining of HIF-1a was obtained, by red fluorescence imaging after addition of LOX-1 to medium at a final concentration of 50 µM, with the monolayer SCC-7 cells incubated 24 hours in 20 to 2.5% O2 concentration.
(Zhang et al., Cancer Research 2010)

3D: Intra-spheroid hypoxia sensing

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Fluorescence microscopic observation (G-2A Fiter Block: EX510-560, DM575, BA590) was made with various cells, at day 6 of spheroid cultured on NanoCulture Plate, LOX-1 was added into well for 12 hr before imaging. Dense and solid spheroids such as HT-29 and BT474 express high level of hypoxia, while less cohesive spheroids such as PANC-1 or MIAPaCa-2 express comparatively less hypoxic region inside spheroids.

Characteristics of Hypoxia Probe LOX-1

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Spectral Characteristics
Absorption spectrum is given in green and phosphorescence in red. Absorption and phosphorescence are peaked at 483 and 616 nm, respectively.
(Zhang et al., Cancer Research 2010)

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Hypoxia Sensing Mechanism of Hypoxia Probe LOX-1
At oxygen atmosphere, phosphorescence of Hypoxia Probe LOX-1 is quenched by molecular oxygen. When oxygen content get lower, it emits red phosphorescence.
Data provided by Prof. S. Tobita, Gunma Univ. (Patented)

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Low Cytotoxicity
Four types of cell suspensions were cultured with Hypoxia Probe LOX-1 on NanoCulture Plate. Cytotoxicity test by ATP assay was measured after 7 days of cell culture indicated that Hypoxia Probe LOX-1 does not show cytotoxicity up to 2 μM concentration.