Temozolomide (TMZ) (Sigma, T2577), Doxorubicin (DOX) (Selleckchem, S1208), Tirapazime (TPZ) (Sigma, SML0552) and Image-iT Hypoxia Reagent (Life Technologies, H10498) were dissolved in DMSO at 100, 100, 50 and 1 mM respectively. Image-iT Hypoxia Reagent was used in cell culture at 10 μM, whereas DOX, TPZ and TMZ were used at varying concentrations (the final DMSO concentration used was <0.1%). The CellEvent™ Caspase-3/7 Green Detection Reagent (Thermo, R37111) and the CellROX® Orange Reagent (Thermo, C10443) were used to detect apoptosis and ROS production respectively following supplier instructions.
Image it hypoxia reagent
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Image-iT Hypoxia Reagent is a dye-based reagent designed to visualize and detect hypoxic (low oxygen) conditions in cell culture and tissue samples. The reagent selectively binds to and fluorescently labels cells experiencing hypoxia, allowing researchers to monitor and analyze hypoxic environments.
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Lab products found in correlation
Cellevent caspase 3 7 green detection reagent, by Thermo Fisher Scientific (3 mentions)
Cellrox deep red reagent, by Thermo Fisher Scientific (2 mentions)
Hoechst 33342, by Thermo Fisher Scientific (2 mentions)
Temozolomide, by Merck Group (1 mentions)
Doxorubicin, by Selleck Chemicals (1 mentions)
Cellrox orange reagent, by Thermo Fisher Scientific (1 mentions)
Lsm 800, by Zeiss (1 mentions)
Lactate assay kit, by Solarbio (1 mentions)
Hydroxylamine hydrochloride, by J&K Scientific (1 mentions)
Hydrogen peroxide h2o2 assay kit, by Solarbio (1 mentions)
Rhodamine b isothiocyanate rbitc, by Merck Group (1 mentions)
Live dead cell imaging kit, by Thermo Fisher Scientific (1 mentions)
Pluronic f 127, by Merck Group (1 mentions)
Pe annexin 5 apoptosis detection kit 1, by BD (1 mentions)
6 protocols using image it hypoxia reagent
1
Hypoxia-Induced Apoptosis and ROS Assay
2
Hypoxic Cell Visualization in Microchannels
3
Hypoxia Induction and Visualization in Cells
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Based on our previous publication [27 (link)], we used sodium sulphite to generate hypoxia (shown in Chemical Formula (1).
To visualize hypoxia in cells, Image-iT® Hypoxia reagent (Thermo Fisher Scientific, Waltham, MA, USA) was used with a final concentration of 5 µM. This reagent starts display fluorescence while the oxygen levels decrease in the cells from 5% [27 (link),28 (link)]. HEK cells were cultured in the reservoir on the microfluidic chip and, after 24 h incubation, the medium was replaced with 800 µL of fresh culture media containing 5 µM Image-iT® Hypoxia reagent. The microfluidic chip setup was incubated under the cell culture chamber of Nikon Eclipse Ti-EN- STORM microscope (Nikon, Nikon Instruments Europe BV, Amsterdam, The Netherlands) with 5% CO2 at 100× magnification. The oxygen depleted water started to pump with a flowrate of 0.1 mL/h for 7 h. During pumping, images were recorded from cells with 15 min intervals.
To evaluate the hypoxia response from one or two channels, data were collected from two individual experiments (n = 2).
To visualize hypoxia in cells, Image-iT® Hypoxia reagent (Thermo Fisher Scientific, Waltham, MA, USA) was used with a final concentration of 5 µM. This reagent starts display fluorescence while the oxygen levels decrease in the cells from 5% [27 (link),28 (link)]. HEK cells were cultured in the reservoir on the microfluidic chip and, after 24 h incubation, the medium was replaced with 800 µL of fresh culture media containing 5 µM Image-iT® Hypoxia reagent. The microfluidic chip setup was incubated under the cell culture chamber of Nikon Eclipse Ti-EN- STORM microscope (Nikon, Nikon Instruments Europe BV, Amsterdam, The Netherlands) with 5% CO2 at 100× magnification. The oxygen depleted water started to pump with a flowrate of 0.1 mL/h for 7 h. During pumping, images were recorded from cells with 15 min intervals.
To evaluate the hypoxia response from one or two channels, data were collected from two individual experiments (n = 2).
4
Multimodal Nanoformulation for Hypoxia Monitoring
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PFOB, Pluronic F‐127, Iron (III) chloride hexahydrate (FeCl3 · 6H2O), LOX from Aerococcus viridans, 2′,7′‐Dichlorofluorescin diacetate (DCFH‐DA), IR‐780 iodide, and rhodamine B isothiocyanate (RBITC) were purchased from Sigma Aldrich (USA). Methylene blue (MB) was purchased from TCI (Japan). Hydroxylamine hydrochloride, sodium lactate (NaL), and tannic acid (TA) were purchased from J&K Scientific (China). ATP and 1,10‐phenanthroline were purchased from Aladdin Reagent (China). Hydrogen peroxide (H2O2) assay kit, cell counting KIT‐8 (CCK‐8), ATP assay kit, and lactate assay kit were purchased from Solarbio (China). Live/dead cell imaging kit, image‐iT hypoxia reagent, cellROX deep red reagent, Hoechst 33342, and all cell culture reagents were purchased from ThermoFisher Scientific (USA). PE Annexin V Apoptosis Detection Kit I was purchased from BD Biosciences (USA). All reagents and chemicals were purchased commercially and without further purification.
5
Spheroid Hypoxia Imaging Protocol
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A wide range of spheroid sizes were grown. For HeLa, 200 to 1000 were grown and for LNCaP, 280 to 1100 spheroids were grown. The spheroids were then incubated with 10 of Image-iT Hypoxia Reagent (Invitrogen) and then were incubated for 1 h at 37 °C. The media was then exchanged with fresh medium, DMEM or RPMI depending on cell line. Brightfield images of the spheroids were then taken using the Cytation 1 using a 4 objective. Furthermore, a GFP filter set (Excitation 469/35 nm, Emission 525/39 nm) was used to image the hypoxia reagent (Excitation 488 nm, Emission 520 nm).
6
Oxidative Stress and Hypoxia Analysis of PTFL NPs
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The generation of PTFL NPs induced oxidative stress and hypoxia was detected by Image‐iT Hypoxia Reagent (Invitrogen) and CellROX Deep Red Reagent (Invitrogen). First, 4T1 cells were seeded in 96‐well plates at a density of 5 × 103 cells per well. After 24 h, the culture medium was replaced by a fresh one containing P NP, PL NPs, PTF NPs, or PTFL NPs (120 µg mL−1) with a layer of liquid paraffin coverage to prevent the O2 exchange, then co‐cultured for another 6 h. Subsequently, 4T1 cells in each group were washed with PBS three times and stained by hypoxia and oxidative stress probes. CLSM images were taken to qualitatively analyze the intracellular level of hypoxia and oxidative stress in each group. Quantitative analysis of intracellular ROS and hypoxia was obtained by flow cytometry according to the manufacturer's instructions. The cells without any treatments were set as blank.
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