Breathe new life into hypoxia research Intracellular detection of low oxygen tension in live cells.

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 (Figure 1).

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 “Mimic life in three dimensions” on page 20). 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, 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 can not 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 2).


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” on page 9).

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 3). 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].


Visualize apoptosis in context

Multiplexable Click-iT Plus TUNEL Assays for in situ apoptosis detection.

The late stages of apoptosis are characterized by changes in nuclear morphology, chromatin condensation, nuclear envelope degradation, and ultimately fragmentation of cellular DNA. While DNA-binding dyes (e.g., Hoechst™ 33342 and DAPI) are typically used to monitor nuclear morphology and chromatin condensation, DNA fragmentation is routinely detected in situ with the terminal deoxynucleotidyl transferase–dUTP nick end labeling (TUNEL) assay. Here we describe the limitations of conventional TUNEL assays and introduce the Click-iT™ Plus TUNEL Assays for In Situ Apoptosis Detection. Click-iT Plus technology enables you to perform specific and sensitive TUNEL assays that can be multiplexed with other fluorescence-based cell function assays, including those that incorporate fluorescent proteins and a variety of dyes.

The TUNEL assay…then

Since its introduction in 1992, the TUNEL assay has been widely used for the in situ detection of apoptosis. The TUNEL assay is based on the incorporation of modified thymidine analogs by the enzyme terminal deoxynucleotidyl transferase (TdT) at the 3/-OH ends of fragmented DNA. The modification on the nucleotide can be as simple as a bromine atom (5-bromo-2/-deoxyuridine triphosphate or BrdUTP) or a more complex molecule such as a fluorophore or hapten (e.g., biotin-dUTP). Incorporated BrdU is typically detected with an anti-BrdU antibody, followed by a secondary detection reagent. A fluorescently modified nucleotide (e.g., fluorescein-dUTP) can be detected directly, whereas biotin is detected indirectly by the addition of a fluorescent streptavidin conjugate.

The extensive fixation and permeabilization required to give antibodies access to the incorporated BrdU can erase important physical and antigenic characteristics of the tissue being examined. Nonspecific background issues associated with the use of biotin–streptavidin detection systems make fluorescence-based TUNEL assays preferable. However, the size of the fluorophores used to modify the nucleotides can cause a reduction in incorporation rates, decreasing the sensitivity of the TUNEL assay. Additionally, the fluorophores used in most currently available TUNEL assays suffer from high rates of photobleaching, further reducing the assay sensitivity, and they often exhibit significant spectral overlap with other fluorescent dyes and proteins, limiting the ability to multiplex with other fluorescence-based probes.

And now…Click-iT Plus TUNEL Assays

The Click-iT Plus TUNEL Assays for In Situ Apoptosis Detection were developed to address the issues affecting sensitivity, photobleaching, and multiplexability. These assays use an EdUTP (a dUTP nucleotide modified with a small alkyne moiety), which is incorporated at the 3/-OH ends of fragmented DNA by the TdT enzyme. Detection is based on a highly specific click reaction, a copper-catalyzed covalent reaction between a fluorescent Alexa Fluor™ picolyl azide dye and the alkyne moiety on the EdUTP. Because of the small size of the alkyne moiety, the EdUTP nucleotide is more readily incorporated by TdT than other modified nucleotides. In addition, the small size of the Alexa Fluor picolyl azide allows easier access to the incorporated nucleotides, negating the need for harsh DNA denaturing techniques required for antibody-based detection of the nucleotide. Moreover, the Alexa Fluor picolyl azide dye serves as a brightly fluorescent and photostable detection reagent and is available in three emission colors for flexibility in multiplex assays.

The Click-iT Plus technology improves upon the first-generation Click-iT TUNEL Assays by the use of the picolyl azide combined with a copper protectant to reduce the exposure of fluorescent proteins, such as Green Fluorescent Protein (GFP) or Red Fluorescent Protein (RFP), to the damaging effects of free copper. In addition, standard aldehyde-based fixation and detergent permeabilization are sufficient for the Click-iT Plus EdU detection reagent to gain access to the DNA; no harsh denaturants are required. The gentle reaction and detection conditions of the Click-iT Plus TUNEL Assay enable you to multiplex this assay with fluorescent proteins, labeled phalloidins, and other copper-sensitive fluorophores (Figure 1).


Specific, multiplexable TUNEL assays

To demonstrate the specificity of the Click-iT Plus TUNEL Assay in a variety of cell types, four formalin-fixed, paraffin-embedded (FFPE) tissue sections were obtained. The mouse colon, heart, liver, and intestine tissue sections were deparaffinized, fixed, and permeabilized. As a substitute for the DNA nicking that occurs in late-stage apoptosis, the tissue sections were treated with DNase I. Tissue sections without DNase I treatment were used as controls. After treatment, the nicked DNA representing the apoptotic signal was detected using the Click-iT Plus TUNEL Assay with Alexa Fluor 594 dye; no significant fluorescence was detected in the control samples (Figure 2).


To demonstrate the ability to multiplex with the Click-iT Plus TUNEL Assays, we used FFPE intestine tissue sections from a transgenic mouse in which GFP expression was localized to the muscularis externa (muscular layer surrounding the intestine). The tissue sections were subjected to deparaffinization, permeabilization, and DNase I treatment. After treatment with the Click-iT Plus TUNEL Assay (Alexa Fluor 594 dye), the tissue sections were stained with Alexa Fluor 647 dye–conjugated phalloidin and Hoechst 33342 dye. Figure 3 clearly shows that all four fluorescent signals—fragmented DNA labeled with Alexa Fluor 594 dye, muscularis externa expressing GFP, nuclei labeled with Hoechst 33342 dye, and actin staining with Alexa Fluor 647 phalloidin—can be visualized in a single tissue section.


Choose from three fluorescent colors

The Click-iT Plus TUNEL Assays for In Situ Apoptosis Detection are available with detection reagents that fluoresce green (Alexa Fluor 488 picolyl azide), red (Alexa Fluor 594 picolyl azide), or deep red (Alexa Fluor 647 picolyl azide). These assays have been optimized and contain all the components necessary to label and detect apoptotic cells from FFPE tissue sections or adherent cells grown on coverslips. The kits include sufficient reagents for labeling 50 coverslips (18 x 18 mm) and can be configured for 50 independent TUNEL apoptosis tests. To learn more, go to

Click-iT™ Plus TUNEL Assay for In Situ Apoptosis Detection, Alexa Fluor™ 488 dye 1 kit C10617
Click-iT™ Plus TUNEL Assay for In Situ Apoptosis Detection, Alexa Fluor™ 594 dye 1 kit C10618
Click-iT™ Plus TUNEL Assay for In Situ Apoptosis Detection, Alexa Fluor™ 647 dye 1 kit C10619




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