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Induction and Inhibition Studies of Hypoxia and Oxidative Stress in Immortalized KeratinocytesDownload
June 13, 2013
Using traditional microplate detection and automated digital widefield fluorescence microscopy in the Cytation™3 Cell Imaging Multi-Mode Reader to enable rich phenotypic data generation and rapid screening of inhibitors
Authors: Brad Larson and Peter Banks, Applications Department, BioTek Instruments, Inc., Winooski, VT; Stephanie Georgiou, Enzo Life Sciences, Farmingdale, NY
We have demonstrated a multiplexed assay that can monitor the induction of hypoxia and oxidative stress phenotypes using fluorogenic probes. Data readout can be achieved using whole well fluorescence intensities measured using either monochromators or spectral filters for rapid measurements depicting relative phenotype changes. Absolute ratios of effected cells for each phenotype can also be obtained through cellular analysis using automated digital widefield fluorescence microscopy. We then assessed the ability of a library of antioxidants for their ability to inhibit hypoxia in a separate assay. The library was screened rapidly using whole well intensities. A hit picking feature in the instrument software was activated to image only those wells meeting the predetermined criteria. Imaging then provided qualitative and quantitative assessments of the extent of hypoxia phenotype inhibition. Excessive inhibition relative to controls was found to be caused by compound toxicity using a luminescent cell viability assay. All cell-based assays were performed using the Cytation™3 Cell Imaging Multi-Mode Reader.
Hypoxia is a pathological condition where the entire body, or a portion of the body, is deprived of adequate oxygen supply. Variations in oxygen concentration can be part of normal physiology; such as during strenuous exercise, high altitude climbs, or deep sea dives. However, hypoxia can also be a serious condition. Many children born prematurely experience generalized hypoxia due to the fact that their lungs have not fully developed, and oxygenated blood is not adequately distributed throughout the body.
Hypoxic skin injuries are also an important pathological process in many diseases, including multiple types of ulcer such as pressure, diabetic and varicose ulcers [1,2,3]. Insufficient blood or oxygen supply is a leading causal factor, and can lead to chronic, non-healing ulcers [4,5,6]. It has been shown that oxidative stress, the overproduction of reactive oxygen species (ROS), is intimately associated with hypoxic injury of skin . This has led to additional studies  which have examined the potential protective ability of antioxidants against hypoxia and its downstream effects.
In this application note we demonstrate an in vitro multiplexed microplate assay that can monitor induction of hypoxia and oxidative stress through ROS production. Cobalt Chloride (CoCl2), a well-known mimetic agent of hypoxia , was used to chemically induce these phenotypes in immortalized keratinocytes. Relative whole well fluorescence intensity data were acquired for both assays using PMT-based fluorescence microplate detection, in addition to using automated digital wide-field fluorescence microscopy. The latter provides quantitative ratios of affected cells relative to the total cell population using the cellular analysis feature of Gen5 software in addition to qualitative visual confirmation of oxidative stress and hypoxia.
A screen of documented antioxidant compounds was performed to determine whether induction of the hypoxic condition could be inhibited using the cell model included here. A hit picking feature of the instrument’s software allowed for rapid screening of compounds using whole well intensities followed by imaging of only hit wells to collect phenotypic data.
Dose response tests were then performed in order to confirm the effects seen from select inhibitor compounds. Finally, a luminescent cell viability assay was incorporated where cell viability measurements were performed in the same well following detection of the fluorescent signals from the Hypoxia/Oxidative Stress assay. All measurements were performed on the Cytation™3 Cell Imaging Multi-Mode Reader.
Materials and Methods
Immortalized transformed keratinocytes (Catalog No. CRL-2309) were obtained from American Type Culture Collection (ATCC) (Manassas, VA). The cells were propagated in Keratinocyte Serum Free Medium (Catalog No. 17005-042) supplemented with Bovine Pituitary Extract (Catalog No. 13028-014) and EGF Recombinant Human Protein (Catalog No. PHG0311) from Life Technologies (Carlsbad, CA).
Hypoxia/Oxidative Stress Detection Kit (Catalog No. ENZ-51042-K100) was donated by Enzo Life Sciences (Farmingdale, NY). CellTiter-Glo® Luminescent Cell Viability Assay (Catalog No. G7572) was purchased from Promega Corporation (Madison, WI). BisBenzimide H33342 trihydrochloride (Hoechst 33342) (Catalog No. 14533) was purchased from Sigma-Aldrich (Saint Louis, MO).
Cobalt(II) Chloride, hexahydrate (Catalog No. C2911) from Sigma-Aldrich (Saint Louis, MO) was used to chemically induce hypoxia in the keratinocytes.
N-acetyl-L-cysteine (NAC) (Catalog No. A7250), was purchased from Sigma-Aldrich (Saint Louis, MO). The Screen-Well® REDOX Library, V.1.1 (Catalog No. BML-2835-0100) was donated by Enzo Life Sciences (Farmingdale, NY).
96-Well Flat Clear Bottom, Black PS, TC-Treated Microplates (Catalog No. 3904) were purchased from Corning Life Sciences (Corning, NY).
Cytation™3 combines automated digital widefield microscopy and conventional microplate detection. This patent pending design provides rich phenotypic cellular information with well-based quantitative data. Equipped with BioTek’s patented Hybrid Technology™ for microplate detection, Cytation3 includes both high sensitivity filter-based detection and a flexible monochromator based system for unmatched versatility and performance. The upgradable automated digital fluorescence microscopy module provides researchers rich cellular visualization analysis without the complexity and expense of standard microplate-based imagers.
The filter-based system was used to detect the green fluorescent signal from the Oxidative Stress Detection Reagent with the following settings: 485/20 nm Excitation Filter; 528/20 nm Emission Filter; 510 nm Cutoff Mirror; Delay after plate movement: 100 msec; Read height: 4.5 mm. The monochromator-based system was used to detect the red fluorescent signal from the Hypoxia Detection Reagent with the following settings: 540 nm Excitation; 605 nm Emission; Delay after plate movement: 100 msec; Read height: 4.5 mm.
The luminescent detection system was used to detect the luminescent signal from the CellTiter-Glo® Assay using the following settings: Delay after plate movement: 100 msec; Integration Time: 0.3 sec; Read height: 4.5 mm. 20X imaging was then performed with the Hypoxia/Oxidative Stress multiplexed assay and Hoescht 33342 fluorescent probe using the microscopy capabilities. Gen5™ software was used for initial data analysis.
Hypoxia/Oxidative Stress Detection Kit
The Hypoxia/Oxidative Stress Detection Kit from Enzo Life Sciences (Farmingdale, NY) is designed for functional detection of hypoxia and oxidative stress levels in live cells (both suspension and adherent). The kit includes fluorogenic probes for hypoxia (red) and for oxidative stress levels (green) as two major components. Red Hypoxia Detection probe is a non-fluorescent or weakly fluorescent aromatic compound containing a nitro (NO2) moiety. Due to increased nitroreductase activity in hypoxic cells, the nitro group is converted in a series of chemical steps to hydroxamino (NHOH) and amino (NH2) group. The original molecule then degrades releasing the fluorescent probe. Oxidative Stress Detection Reagent is a non-fluorescent, cell-permeable total ROS detection dye which reacts directly with a wide range of reactive species yielding a green fluorescent product indicative of cellular production of different ROS types.
The assay protocol described here was created following inital optimization. Keratinocytes, at a concentration of 1.0x105 cells/mL, were added to the 96-well cell plates in a volume of 100 μL and incubated overnight. For the agonist protocol, 50 μL of 3x cobalt chloride (CoCl2) was then added to the well and incubated at 37 °C/5% CO2 for the appropriate time. For the inhibitor protocol, 25 μL of 6x inhibitor was added to the well and incubated for 60 minutes at 37 °C/5% CO2. 25 μL of 6x CoCl2 was then added to the well and incubated for 2 hours using the same conditions. Following incubation the medium was removed, and the plate was washed once with 100 μL of 1x PBS. 50 μL of PBS containing the Hypoxia, Oxidative Stress, and Hoechst 33342 fluorescent probes was then added to the wells and incubated at 37 °C/5% CO2 for 30 minutes. Upon completion the plate was washed three times with 100 μL PBS, and a final volume of 50 μL PBS was added to the wells before the plate reads and imaging were performed.
Hypoxia/ROS Stimulation Optimization
Initial experiments were performed to determine whether CoCl2 could induce hypoxia in the cell model being used, as well as the ability of the Cytation™3 to detect the fluorescent signals from the fluorogenic probes for both Hypoxia (red) and Oxidative Stress (green) using conventional microplate detection. In the first experiment, 400 μM CoCl2 (1x) was added to the cells and incubated using the same conditions between 30 minutes and 5 hours. In the second, six different concentrations of CoCl2 were added to the cells, ranging from 0-1000 μM (1x), and incubated at 37 °C/5% CO2 for 2 hours. Microplate reads were performed on all wells for each experiment as well as 20x imaging of the various [CoCl2]s of the dose response.
REDOX Compound Library Screen
The 84 member REDOX compound library was then evaluated to determine if inhibitors of chemically induced hypoxia could be identified. A single well of each compound was tested at a final 1x concentration of 10 μM. Four individual concentrations of the known ROS scavenger, NAC were also included along with uninhibited positive control and uninduced negative control wells. A final 1x concentration of 500 μM CoCl2 was added to test and positive control wells.
Gen5™ Microplate Reader/Imager Hypoxia Inhibition Hit Pick Protocol
A single protocol was created with the Gen5 software for use during the compound library screen. The protocol eliminates the need to image the entire cell plate, therefore obviating unnecessary data generation and storage. The plate layout created in Gen5 identifies the location of control and test wells (Figure 1).
Figure 1. Gen5 plate layout for Enzo REDOX Compound Library Screen.
The Hit Pick procedure calls for all wells of the plate to be read using the Hypoxia assay read parameters (Figure 2A).
Those wells with an RFU value greater than one standard deviation lower than the average from the four positive control wells, which corresponds to greater than or equal to 50% inhibition, are then imaged (Figure 2B).
Figure 2. (A) Hit Pick microplate read and imaging procedure. (B) Wells imaged using Hit Pick procedure criteria.
Select Compound Inhibition/Cytotoxicity Analysis
Seven compounds plus NAC were selected for further analysis to determine the complete inhibitory profile and cytotoxic potential of each. 10 concentrations of inhibitor were tested, ranging from 0-100 μM for library compounds and 0-10 mM for NAC, plus uninhibited positive control and uninduced negative control wells as included with the library screen. A final 1x concentration of 500 μM CoCl2 was once again added to test and positive control wells. The cell wash and probe addition process were performed as previously described. Following reading of the Hypoxia/Oxidative Stress assays and 20x imaging, an equal volume (50 μL) of CellTiter-Glo® reagent was added to the wells. The plate was shaken for 30 seconds and incubated for 10 minutes at room temperature. The luminescent signal was then detected from each well to assess cell's viability.
Results and Discussion
Hypoxia/Oxidative Stress Detection and Optimization
The results generated from the first experiment using 400 μM CoCl2 concentrations and multiple incubation times (Figure 3A) demonstrate that CoCl2 induces hypoxia in the immortalized keratinocytes used here, similar to what has been shown with other keratinocyte cell models . Furthermore, the similarity in the tracking of the delta RFU values seen from the Oxidative Stress assay (Figure 3B) confirms the role that overproduction of ROS plays in the creation of a hypoxic condition within the cells. It can also be seen that a 2 hour incubation time creates the largest change in fluorescence from either assay.
Figure 3. Evaluation of incubation time effect using 400 μM CoCl2 for (A) Hypoxia and (B) Oxidative Stress assays.
When variable concentrations of CoCl2 are added to the keratinocytes and incubated for 2 hours (Figure 4), it is apparent that the 500 μM concentration, while not causing the greatest change in RFU values, still causes a significant hypoxic effect in the cells.
Figure 4. Evaluation of CoCl2 dose response using 2 hour incubation for (A) Hypoxia and (B) Oxidative Stress assays.
This is also verified qualitatively when examining the 20x images captured in the second experiment (Figure 5).
Figure 5. 20x Cytation™3 combined images of Hoechst 33342 (blue), Hypoxia (red), and Oxidative Stress (green) probes following 2 hour CoCl2 incubation.
Figure 6. Cellular Analysis images of (A) Hoechst 33342; (B) Oxidative Stress; and (C) Hypoxia stained cells. Analysis parameters included Threshold: 10,000 RFU; Minimum Object Size: 10 μm; and Maximum Object Size: 150 μm. (D) Ratio of effected to total cell number for increasing CoCl2 concentrations.
From the microplate read and 20x imaging results it was decided that a 500 μM CoCl2 concentration and 2 hour incubation time would be used for subsequent inhibitor experiments of hypoxia as this concentration yielded a change in well fluorescence intensity of ~ 15,000 RFU and ~ 75% of cells affected.
Enzo Screen-Well® REDOX Library Screen
The Screen-Well REDOX library, which contains a number of known antioxidant compounds, was screened for inhibition of CoCl2 induced-hypoxia. Since the two phenotypes of oxidative stress and hypoxia were strongly linked, only hypoxia was assessed in the screen.
A number of antioxidant compounds were identified as being able to inhibit CoCl2 induced hypoxia by ≥50% (Figure 7). As previously mentioned, the Hit Pick feature in the Gen5 software allowed for imaging of wells containing only “Hit” compounds plus identified control wells (Figure 2B), eliminating needless data creation and storage. Several of these compounds, along with the positive control compound, NAC, were carried forward for dose response tests to determine their exact inhibitory characteristics and potential cytotoxic effects.
Figure 7. (A) Screen-Well REDOX Library screen percent inhibition results calculated from whole well microplate reads using the following formula: (1-((RFU Value(Test Well) – RFU Value(Neg Ctl))/(RFU Value(Pos Ctl) – RFU Value(Neg Ctl))))*100. (B) 20x images from select wells exhibiting ≥50% inhibition, and no compound control, using Hit Picking feature in Gen5.
Select Compound Inhibition/Cytotoxicity Confirmation
Dose response curves for the seven library compounds chosen, plus NAC were plotted using the percent inhibition values calculated for each concentration tested (Figure 8). From the results it can be seen that hypoxia is fully inhibited by each compound at the highest concentrations. Indeed excessive inhibition is observed for a number of the compounds tested, as evidenced by Gossypol (Figure 7B).
Figure 8. Complete dose response curves for selected library inhibitor compounds and NAC.
Effect on keratinocyte cell viability was also assessed across the full compound dose range.
Figure 9. (A) % cell viability values for selected inhibitory compounds. 20x images also shown for (B) well containing 500 μM CoCl2 plus no inhibitor; (C) inhibition via NAC of CoCl2 induced hypoxia with no effect on cell viability; and (D) disulfiram inhibition due to significant loss of cell viability.
From the cell viability data generated using the CellTiter- Glo® assay, and 20x images captured (Figure 9), it is apparent that high concentrations of most compounds tested demonstrate significant cytotoxic effects on the keratinocyte cells. Therefore a decreased fluorescent signal from the Hypoxia assay cannot always be attributed to true inhibition of the CoCl2 induced effects.
These results are in keeping with previously published findings illustrating the fact that exogenous antioxidants are beneficial at physiological concentrations, but can have detrimental effects at high concentrations .
The Hypoxia/Oxidative Stress Detection kit provides an easy-to-use, multiplexed cell-based approach for the assessment of hypoxia induction and ROS creation. Upon the addition of an endpoint luminescent cell viability reagent, a triplexed method is created which also allows for the detection of potential cytotoxicity. Optical paths can be used to collect whole well fluorescent and luminescent intensities. In this work we used spectral filters for the assay quantifying the oxidative stress phenotype as the spectral characteristics of the probe are equivalent to fluorescein, which is a filter set that comes standard with all multi-mode readers. The spectral characteristics of the probe for the hypoxia phenotype is much less common, thus we used the inherent wavelength flexibility and fast reading speed of monochromators for this dye. Finally, the dedicated luminescence detection system is used to quantify the signal from the cell viability reagent.
The two probes can also be used with fluorescence microscopy. The automated digital widefield fluorescence module of Cytation3, in addition to the cell segmentation capabilities of Gen5 software, provides rich phenotypic data including qualitative visual confirmation of cells affected by the phenotypes and absolute quantification of the ratio of cells affected by the phenotype relative to total cell populations.
For screening of inhibition of phenotypes, whole well intensities can be used for rapid screening of plates and only those wells which display reduction of signal below a chosen threshold need be imaged for hit confirmation and quantification of phenotype. This serves to dramatically speed up screening compared to using the digital widefield microscopy alone common to some of the less expensive HCS instruments and relieve issues with data storage. Compared to typical multi-mode readers, i.e. those without automated digital fluorescence microscopy, Cytation3 provides much richer phenotypic data and confirmatory results.
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