Breathe new life into hypoxia research

(See a list of the products featured in this article.)

Oxygen homeostasis is an important physiological process that is required to maintain cellular health and function. Hypoxia is a condition of low oxygen tension in tissues and contributes significantly to the pathophysiology of major categories of human disease, including myocardial and cerebral ischemia, cancer, pulmonary hypertension, congenital heart disease, and chronic obstructive pulmonary disease. While generally associated with pathological conditions, hypoxia response pathways are also critical in the normal development of some cell types, such as hematopoietic stem cells. Although the significance of hypoxia in biological processes is well known, creating model systems to accurately control hypoxic conditions is extremely difficult for most researchers without access to elaborate instruments that allow precise control and maintenance of temperature, humidity, and gases (CO2 and O2) during an experiment. Fortunately, the EVOS FL Auto Imaging System with Onstage Incubator provides an easy-to-use platform that allows for the precise control of oxygen levels, thereby delivering an effective system for researchers to evaluate cellular responses to hypoxia by live-cell fluorescence imaging using the Image-iT Hypoxia Reagent.

Cellular responses to hypoxia

The growth patterns of solid tumors impose low oxygen concentrations on their core cells, and therefore adaptation to hypoxia is advantageous for tumor development and survival [1]. Conversely, disruption of these hypoxic responses can lead to leukemic transformation [2]. Hypoxic responses are also critical for the normal development of hematopoietic stem cells, which reside within a hypoxic bone marrow microenvironment. Hypoxia signaling pathways are used for cell fate decisions leading to normal hematopoiesis [3].

Adaptation to hypoxia is mediated largely by transcriptional activation of genes that facilitate short-term (e.g., glucose transport) and long-term (e.g., angiogenesis) adaptive mechanisms. A key regulator of cellular responses to hypoxic conditions is the transcription factor HIF-1 (hypoxia inducible factor-1), which functions as a master regulator of cellular and systemic homeostatic response to hypoxia by activating transcription of a wide array of genes, including those involved in energy metabolism, angiogenesis, and erythropoiesis, as well as genes whose protein products increase oxygen delivery or facilitate metabolic adaptation to hypoxia.

Methods for studying hypoxia

While the importance of studying the cellular signaling pathways involved in the hypoxic response is clearly understood in a wide range of biological applications, developing model systems to precisely study the effects of low oxygen levels on cells and tissues remains technically challenging for most laboratories. One common method for studying the downstream effects of HIF-1 depletion involves the addition of cobalt chloride (CoCl2) to cells in culture. CoCl2 can mimic the effects of hypoxia by stabilizing the HIF-1 complex and thereby activating HIF-1–inducible genes. However, CoCl2 only impacts the HIF-1 pathway and may not affect other hypoxia-related pathways. In addition, other unknown cellular processes and functions may be disrupted by CoCl2 treatment, inducing phenotypes that are unrelated to the hypoxic response. Techniques for imaging hypoxia include the use of invasive oxygen electrodes to measure tissue oxygen levels, HIF-1 or Glut1 tissue stains to look for indirect evidence of hypoxia in cells, and nitroimidazoles that bind to protein thiols in hypoxic tissue at acute reductions in oxygen levels.

Spheroid culture methods have enabled substantial contributions to both basic cell biology and cancer biology (see “Formation of uniform and reproducible 3D cancer spheroids in high-throughput plates"). The multicellular tumor spheroid (MCTS)—a 3D cell structure with a diameter of 200–500 μm—is a valuable model for cancer biology. Closely mimicking the physiology of small avascular tumors [4], spheroids in this model develop chemical gradients of oxygen, nutrients, and catabolites just like a tumor in vivo; they also possess histomorphological and functional features similar to those of tumors.

Both spheroids and tumors exhibit a heterogeneous distribution of cell types, expression patterns, and physiology. Cells located at the surface of a spheroid secrete specific compounds as a tumor would in vivo. Internally, spheroids possess the same hypoxic core seen in tumors; this hypoxic core is one of the most distinct characteristics of spheroid cultures that cannot be successfully reproduced with classic 2D culture methods. The MCTS model thus mimics in vivo solid tumors in which cells rapidly outgrow the blood supply, leaving the center of the tumor with an extremely low oxygen concentration (Figure 1).

Figure 1. Tumor spheroids visualized with the Image-iT Hypoxia Reagent. (A, B) HeLa cells were grown on Thermo Scientific Nunclon Sphera 96-well U-bottom plates for 2 days in complete medium to allow for spheroid formation. Spheroids were stained with 5 μM Image-iT Hypoxia Reagent (red) for 3 hr. NucBlue Live ReadyProbes Reagent (blue) was used as nuclear counterstain. Images were acquired on an EVOS FL Auto Imaging System. (C) HeLa cells were grown in complete medium on NanoCulture Plates (Scivax Life Sciences) for 6 days to allow for spheroid formation, and then spheroids were stained with 10 μM Image-iT Hypoxia Reagent for 1 hr. Images were acquired on a Zeiss™ LSM 710 confocal microscope. Hypoxic conditions at the core of the spheroids are represented by red staining.

The EVOS FL Auto Imaging System with Onstage Incubator: Live-cell imaging under precisely controlled oxygen levels

Incubation chambers have been used to lower oxygen concentrations to allow for long-term cell growth in hypoxic conditions. However, real-time visualization of cellular processes in response to hypoxic conditions becomes problematic when transferring cells from the incubator to a microscope for imaging. Not only is it difficult to achieve precise control of oxygen levels in an incubator, but reoxygenation may create misleading results during the time required to image hypoxic cells.

The EVOS FL Auto Imaging System with Onstage Incubator includes an environmental chamber allowing for the precise control of oxygen levels, temperature, and humidity, thereby delivering an effective system for researchers to evaluate cellular responses to hypoxia over long time periods by live-cell fluorescence imaging. The onstage incubator contains port connections for air, O2, and N2. Gas concentrations are controlled by the software on the EVOS FL Auto system, allowing cells to be cultured using precise O2 concentrations over an extended period of time. The EVOS FL Auto Imaging System with Onstage Incubator is easy to use: Simply input the desired O2 level and the onstage incubator will equilibrate to the selected conditions. The incubator is designed specifically for the EVOS automated imaging system, which combines live-cell imaging, area scanning, image stitching, and time-lapse imaging in a single user-friendly platform. EVOS imaging systems make multichannel fluorescence microscopy accessible to both novice users and high-throughput core imaging facilities (see “The EVOS FL Cell Imaging System: A key component of an imaging core facility").

Image-iT Hypoxia Reagent: A real-time oxygen detector

With the EVOS FL Auto Imaging System, live-cell imaging can be performed in real time under hypoxic conditions using the Image-iT Hypoxia Reagent. The Image-iT Hypoxia Reagent is a fluorogenic, cell-permeant compound for measuring hypoxia in live cells. This reagent is nonfluorescent in an environment with normal oxygen concentrations (approximately 20%) and becomes increasingly fluorescent as oxygen levels are decreased (Figure 2). Unlike nitroimidazoles (such as pimonidazole) that respond only to very low oxygen levels (<1%) [5,6], the Image-iT Hypoxia Reagent begins to fluoresce when oxygen levels drop below 5%.

Because it responds quickly to a changing environment, Image-iT Hypoxia Reagent can serve as a real-time oxygen detector, with a fluorescent signal that increases as atmospheric oxygen levels drop below 5% and decreases if oxygen concentrations increase. In addition, Image-iT Hypoxia Reagent is very easy to use; just add it to cell culture medium and image. These properties make this reagent an ideal tool for detecting hypoxic conditions in tumor cells, 3D cultures, spheroids, neurons, and other tissues used in hypoxia research. Reagents with similar applications have been reported to detect tumors in small animals, and their fluorescence properties have been shown to correspond with increased HIF-1α expression and translocation in hypoxic environments [7].

Figure 2. Imaging hypoxia with the Image-iT Hypoxia Reagent using the EVOS FL Auto Imaging System with Onstage Incubator. A549 cells were labeled with Image-iT Hypoxia Reagent (red) to visualize the cellular response to changing oxygen levels, and NucBlue Live ReadyProbes Reagent (blue) was used to label nuclei in all cells. (A) Under normal conditions (20% O2), the Image-iT Hypoxia Reagent is nonfluorescent. Fluorescence increases as oxygen levels are decreased to (B) 5% O2, (C) 2.5% O2, and (D) 1% O2. The NucBlue Live ReadyProbes Reagent, a formulation of Hoechst 33342, is used as a counterstain for autofocusing throughout the experiment; its fluorescence remains relatively unchanged under hypoxic conditions. (E) The user interface of the EVOS FL Auto Imaging System with Onstage Incubator shows the environmental chamber setup.

Immunodetection of HIF-1 in fixed cells

In fixed-cell imaging, HIF-1 expression is commonly used as a marker for monitoring cells following exposure to hypoxic conditions. The HIF-1α mouse monoclonal antibody provides a highly specific probe for evaluating HIF-1α expression in fixed-cell samples. We offer a number of Thermo Scientific Pierce antibodies that recognize HIF-1α and have been verified to perform in various applications, including immunocytochemistry (ICC), immunohistochemistry (IHC), western blot analysis, and immunoprecipitation; Figure 3 shows an example of ICC using the anti–HIF-1α mouse monoclonal antibody, clone mgc3, which has citations describing its use in all of the above applications. Also available is the well-validated anti–HIF-1α mouse monoclonal antibody, clone H1alpha67, which has proven useful in at least 10 different types of immunoassays, including western blotting (Figure 4).

Figure 3. Immunofluorescence analysis of HIF-1α localization in HeLa cells after treatment with desferrioxamine. HeLa cells were either left untreated (left panel) or treated with 100 μM desferrioxamine mesylate (a hypoxia-mimetic agent, right panel) for 16 hr. After fixation with formalin, cells were permeabilized with 0.1% Triton X-100 in TBS for 15 min, blocked with 0.3% BSA for 15 min, and labeled with anti–HIF-1α mouse monoclonal antibody (clone mgc3) at a dilution of 1:100 for 1 hr at room temperature. Cells were then washed with PBS, and incubated with a DyLight 488 goat anti–mouse IgG (H+L) secondary antibody (green) at a dilution of 1:500 for 30 min at room temperature. F-actin was stained with DyLight 594 Phalloidin (red), and nuclei were stained with Hoechst 33342 (blue). Images were taken on a Thermo Scientific ArrayScan instrument at 20x magnification.

Figure 4. Western blot analysis of HIF-1α localization in COS-7 nuclear extracts after treatment with cobalt chloride. COS-7 cells were treated with cobalt chloride (CoCl2), which stabilizes the HIF-1 complex, mimicking the effects of hypoxia. Nuclear extracts were prepared and subjected to western blot analysis using anti–HIF-1α mouse monoclonal antibody (clone H1alpha67).

Accelerating the pace of hypoxia research

The EVOS FL Auto Imaging System with Onstage Incubator allows for the precise control of oxygen concentrations, overcoming technical hurdles that have plagued hypoxia research in the past. This easy-to- use system, in combination with the Image-iT Hypoxia Reagent, is ideal for studying the role of cellular responses to hypoxia in both basic biological processes and disease mechanisms.


  1. Giaccia AJ, Simon MC, Johnson R (2004) The biology of hypoxia: the role of oxygen sensing in development, normal function, and disease. Genes Dev 18:2183–2194.
  2. Gezer D, Vukovic M, Soga T et al. (2014) Concise review: genetic dissection of hypoxia signaling pathways in normal and leukemic stem cells. Stem Cells 32:1390–1397.
  3. Rankin EB, Giaccia AJ, Schipani E (2011) A central role for hypoxic signaling in cartilage, bone, and hematopoiesis. Curr Osteoporos Rep 9:46–52.
  4. Kunz-Schughart LA (1999) Multicellular tumor spheroids: intermediates between monolayer culture and in vivo tumor. Cell Biol Int 23:157–161.
  5. Arteel GE, Thurman RG, Yates JM et al. (1995) Evidence that hypoxia markers detect oxygen gradients in liver: pimonidazole and retrograde perfusion of rat liver. Br J Cancer 72:889–895.
  6. Gross MW, Karbach U, Groebe K et al. (1995) Calibration of misonidazole labeling by simultaneous measurement of oxygen tension and labeling density in multicellular spheroids. Int J Cancer 61:567–573.
  7. Zhang S, Hosaka M, Yoshihara T et al. (2010) Phosphorescent light-emitting iridium complexes serve as a hypoxia-sensing probe for tumor imaging in living animals. Cancer Res 70:4490–4498.


Learn more about


Article download

Download a hyperlink-enabled, printer-friendly version of this article.

Download now