Cellular responses to reduced oxygen—hypoxic conditions—have been linked to a wide range of human pathologies, including tumor development, atherosclerosis, inflammation, and abnormal angiogenesis. Although the importance of hypoxia in inducing these conditions is well known, creating model systems to accurately control the hypoxic conditions is extremely difficult for most researchers without access to elaborate systems that allow maintenance and precise control of temperature, humidity, and gases (CO2 and O2) during an experiment. The Invitrogen™ EVOS™ FL Auto Imaging System with Onstage Incubator provides an environmental chamber allowing for the precise control of oxygen levels, thereby delivering an effective system for researchers to evaluate cellular responses to hypoxia by long-term fluorescence live-cell imaging. Here we provide step-by-step instructions for setting up hypoxic conditions with the EVOS FL Auto Imaging System with Onstage Incubator.
In this particular study, response to cellular hypoxia was visualized using Invitrogen™ Image-iT™ Hypoxia Reagent. Prior to live-cell imaging, A549 cells were labeled using this fluorescent hypoxia indicator to visualize the cellular response to changing oxygen levels, and Invitrogen™ NucBlue™ Live ReadyProbes™ Reagent was used to label nuclei in all cells. In normal conditions, the Image-iT Hypoxia Reagent is nonfluorescent but increases in fluorescence as oxygen levels are decreased in the cells. The NucBlue reagent, a form of Hoechst 33342, acts as a counterstain that is used for autofocusing throughout the experiment because its fluorescence remains relatively unchanged under hypoxic conditions.
By providing precise control of temperature, oxygen, and humidity levels, the EVOS FL Auto Imaging System with Onstage Incubator delivers a long-term live-cell imaging environment for detailed analysis of cells under hypoxic conditions. In this demonstration, hypoxia was analyzed in A549 cells labeled with Image-iT Hypoxia Reagent and NucBlue Live ReadyProbes Reagent. Under normoxic conditions (20%) there was no signal from the Image-iT Hypoxia Reagent, but in response to the decrease in oxygen levels the signal from the Image-iT Hypoxia Reagent increased, as shown by red punctate staining (Figure 1). Hypoxic cells were first observed at 5% O2 levels, with nearly all the cells being hypoxic at 1% O2 levels. The signal from Image-iT Hypoxia Reagent is reversible: when oxygen levels return to normal, the signal decreases back to baseline (data not shown).
Figure 1. A549 cells stained with Image-iT Hypoxia Reagent and exposed to different oxygen levels. (A) 20% O2. (B) 5% O2. (C) 2.5% O2. (D) 1% O2.
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