This research was supported by the National Institutes of Health CounterACT Program Interagency Agreement# AOD13015-001. We would like to thank Stephanie Froberg and Peter Hurst for their efforts and expertise on video production.

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The authors have nothing to disclose.
DISCLAIMER: The views expressed in this article are those of the author(s) and do not reflect the official policy of the Department of Army, Department of Defense, or the U.S. Government. This research was supported by an interagency agreement between NIH/NIAID and the USAMRICD, and in part by an appointment to the Postgraduate Research Participation Program at the U.S. Army Medical Research Institute of Chemical Defense administered by the Oak Ridge Institute for Science and Education through an interagency agreement between the U.S. Department of Energy and USAMRMC.

Herein we describe our methods and results on the development of a high throughput cornea epithelial cell screening model for the study of HF and CP injuries. We also present the results from the primary siRNA screen for HF injury. There were many challenges to the development of HTS models for the study of TIC injuries. Methods that we could find in the literature related to the study of HF, HFA or CP in cell culture models were of little help. Most in vitro studies on the fluoride ion involve oral cells and ut...

Toxicant-induced ocular injury is a true ocular emergency because chemicals have the potential to rapidly inflict significant tissue damage. Unfortunately, treatments for toxicant-induced corneal injury are only generally supportive as no specific therapeutics exist to treat these injuries. The current treatment strategy is non-specific and primarily includes topical therapeutic treatments such as lubricants, antibiotics, and cycloplegics followed by anti-inflammatories (e.g., steroids) once the cornea has re-epithelialized1,2. Despite the best current therapeutic treatment options available, long-term prognosis is generally poor due to progressive corneal clouding and neovascularization2,3.
Animal models have traditionally been used to investigate chemical toxicity and understand mechanisms of injury. However, animal studies are time consuming and expensive. There are also efforts to reduce animal testing. For example, REACH legislation (EC 1907/2006) in the European Union has provisions intended to reduce animal testing. The provisions include a requirement that companies share data in order to avoid animal testing and obtaining approval from the European Chemicals Agency prior to performing proposed tests on animals. Under the provisions of REACH, animal testing should be a last resort. There is also the European Cosmetics Regulation (EC 1223/2009) that phased out the testing of cosmetics in animals. When animal studies are conducted, they are guided by the principles of 3Rs (Refinement, Reduction, and Replacement), which provide a framework for performing more humane animal research, reducing the number of animals used, and using non-animal alternatives where possible. For these reasons, the field of toxicology has sought to adopt in vitro assays that can provide insight into molecular mechanisms of toxicity and can be done in higher throughput4. This is a functional toxicology approach where toxicants are defined by their function rather than solely by their chemistry. Taken a step further, functional toxicogenomics seek to understand the role(s) that specific genes play in the effects of toxicants5. With the application of siRNA technology, screens to investigate gene function in the molecular and cellular responses to toxicants can be done at high throughput. siRNA are double stranded RNA molecules that are 19-25 nucleotides long that take advantage of the post transcriptional gene silencing pathway present in all mammalian cells6. These are synthetically made and designed to target a specific gene. When introduced into a cell, the siRNA is processed and one strand, the guide strand, is loaded into the RNA-induced silencing complex (RISC). The siRNA directs the RISC to a complementary region in an mRNA molecule, and the RISC degrades the mRNA. This results in the reduction of expression of the specific gene. The resulting reduction of expression of the specific gene can then be studied in toxicant exposed cells to ascertain the function of that gene in the cellular response to the toxicant. Such an approach has been used to further understand the mechanisms of ricin susceptibility and the AHR-dependent induction of CYP1A17,8.
The Chemical Terrorism Risk Assessment (CTRA) list and the toxic industrial chemicals (TIC) listings have itemized select chemicals based on their toxicity and potential to be released during a terrorist, warfare, or industrial accident event9. We are applying an siRNA high throughput screening (HTS) toxicogenomic approach to the study of CTRA list toxicants, which have been identified to be at high risk of use in a terrorist incident. Traditional toxicology seeks to understand the adverse effects that chemicals have on living organisms; however, we have a further desire to understand the mechanisms of injury for the purpose of informing the development of therapeutics and therapeutic approaches, and possibly, to discover molecules which can be targeted for therapeutic development. This effort in some ways may be considered analogous to the use of high throughput siRNA screening and cell based assays in the drug discovery process10. A major difference would be that drug discovery typically seeks a singular target for therapeutic discovery whereas in our approach it is somewhat unlikely that there would be a singular target with high therapeutic value for the treatment of toxicant exposure. We anticipate that any effective treatment paradigm for toxicant exposure would require a multi-faceted approach to achieve high therapeutic value, and toxicogenomic data may vitally inform an effective treatment paradigm.
Benchtop automation brings high throughput methodology to laboratories outside the pharmaceutical or biotech industries. The in vitro studies at our institute have historically been traditional assays which are low throughput11,12,13. In the past few years, our laboratory has transitioned to the use of benchtop robotics to perform high throughput siRNA screening. Herein, we present the refinement of ocular cell models and the development of in vitro exposure methods for hydrogen fluoride (HF) and chloropicrin (CP) suitable for high throughput siRNA screening. Our goal is to identify molecules that regulate cellular injury in response to these toxicants. The targets of the siRNA library we selected include G protein-coupled receptors, protein kinases, proteases, phosphatases, ion channels, and other potentially druggable targets. HF and CP were selected for study by cross-referencing CTRA list agents with the ToxNet reports of industrial accidents to find those that present the greatest risk of ocular injury via vapor exposure9,14. CP (chemical formula Cl3CNO2, CAS number 76-06-2) was originally used as a tear gas in WWI15. It is currently used as an agricultural fumigant and functions as a nematicide, fungicide, and insecticide16. Hydrogen Fluoride (HF) is used in processes including alkylation in oil refineries and electrochemical fluorination of organic compounds17. HF (chemical formula HF, CAS number 62778-11-4) is a gas but in its aqueous form is hydrofluoric acid (HFA, CAS number 7664-39-3). Therefore, we elected to use HFA in our in cell exposure models. The SV40 large T antigen immortalized human corneal epithelial cell line SV40-HCEC was selected for study. Cell viability and the inflammatory marker IL-8 were selected as endpoints because targets that are involved in cellular injury should be reflected in the cell death and the inflammatory response. Specifically, if a target were to play a protective role in toxicant exposure, cell death and/or inflammatory cytokine production should increase when the target expression is inhibited by siRNA. The opposite would be true for targets that play a negative role. Also, chronic inflammation appears to play a role in cornea pathology after exposure, and intervention in cell death pathways may improve clinical outcome2,18.

<table><tbody><tr><td>Bravo liquid handing platform</td><td>Agilent or equivalent</td><td>G5409A</td><td></td></tr><tr><td>Bravo plate shaker</td><td>Agilent or equivalent</td><td>Option 159</td><td></td></tr><tr><td>Bravo 96LT disposable tip head</td><td>Agilent or equivalent</td><td>Option 178</td><td>96-channel large tip pipetting head unit</td></tr><tr><td>Bravo 96ST disposable tip head</td><td>Agilent or equivalent</td><td>Option 177</td><td>96-channel small tip pipetting head unit</td></tr><tr><td>Bravo 384ST disposable tip head</td><td>Agilent or equivalent</td><td>Option 179</td><td>384-channel small tip pipetting head unit</td></tr><tr><td>Bravo 96 250 &amp;mu;L sterile barrier tips</td><td>Agilent or equivalent</td><td>19477-022</td><td></td></tr><tr><td>Bravo 384 30 &amp;mu;L sterile barrier tips</td><td>Agilent or equivalent</td><td>19133-212</td><td></td></tr><tr><td>Bravo 384 70 &amp;mu;L sterile barrier tips</td><td>Agilent or equivalent</td><td>19133-212</td><td></td></tr><tr><td>EnSpire multimode plate reader</td><td>Perkin Elmer or equivalent</td><td>2300-0000</td><td>AlphaLISA assay detector with high power laser excitation</td></tr><tr><td>IL-8 (human) AlphaLISA Detection Kit&amp;nbsp;</td><td>Perkin Elmer or equivalent</td><td>AL224F</td><td>no-wash bead-based assay</td></tr><tr><td>ProxiPlate-384 Plus white 384-shallow well microplates</td><td>Perkin Elmer or equivalent</td><td>6008359</td><td></td></tr><tr><td>Lipofectamine RNAiMAX</td><td>Invitrogen or equivalent</td><td>13778500</td><td>Transfection reagent</td></tr><tr><td>Opti-MEM 1 Reduced Serum Medium</td><td>Invitrogen or equivalent</td><td>31985070</td><td></td></tr><tr><td>TrypLE Express</td><td>Gibco or equivalent</td><td>12605010</td><td>Cell detachment solution</td></tr><tr><td>IncuCyte Zoom</td><td>Essen Instruments or equivalent</td><td>ESSEN BIOSCI 4473</td><td>Incubator-housed automated microscope</td></tr><tr><td>Chloropicrin</td><td>Trinity Manufacturing or equivalent</td><td>N/A</td><td>Acute toxicity and irritant</td></tr><tr><td>DMEM-F12 cell culture medium</td><td>Invitrogen or equivalent</td><td>11330-057</td><td>Contains HEPES</td></tr><tr><td>Fetal bovine serum</td><td>Invitrogen or equivalent</td><td>1891471</td><td></td></tr><tr><td>Human epidermal growth factor (cell culture grade)</td><td>Invitrogen or equivalent</td><td>E9644-.2MG</td><td></td></tr><tr><td>Recombinant human insulin (cell culture grade)</td><td>Invitrogen or equivalent</td><td>12585-014</td><td></td></tr><tr><td>Penicillin-Streptomycin solution (cell culture grade)</td><td>Invitrogen or equivalent</td><td>15140122</td><td></td></tr><tr><td>Hydrocortisone (cell culture grade)</td><td>Sigma or equivalent</td><td>H0888-10G</td><td></td></tr><tr><td>Glucose&amp;nbsp; (cell culture grade)</td><td>Sigma or equivalent</td><td>G7021</td><td></td></tr><tr><td>PBS&amp;nbsp; (cell culture grade)</td><td>Sigma or equivalent</td><td>P5493</td><td></td></tr><tr><td>siRNA</td><td>Dharmacon or equivalent</td><td>various</td><td></td></tr><tr><td>Thiazolyl blue tetrazolium bromide</td><td>Sigma or equivalent</td><td>M5655</td><td>MTT assay substrate</td></tr><tr><td>siRNA buffer</td><td>Thermo or equivalent</td><td>B002000</td><td></td></tr><tr><td>96-well cell culture plates</td><td>Corning or equivalent</td><td>CLS3595</td><td></td></tr><tr><td>T150 cell culture flasks</td><td>Corning or equivalent</td><td>CLS430825</td><td></td></tr><tr><td>BSL-2 cell culture hood</td><td>Nuaire or equivalent</td><td>NU-540</td><td></td></tr><tr><td>300 mL robotic reservoirs</td><td>Thermo or equivalent</td><td>12-565-572&amp;nbsp;</td><td></td></tr><tr><td>96 baffled automation reservoirs</td><td>Thermo or equivalent</td><td>1064-15-8</td><td></td></tr><tr><td>500 mL sterile disposable storage bottles</td><td>Corning or equivalent</td><td>CLS430282</td><td></td></tr><tr><td>Microplate heat sealer</td><td>Thermo or equivalent</td><td>AB-1443A</td><td></td></tr><tr><td>Microplate heat sealing foil</td><td>Thermo or equivalent</td><td>AB-0475</td><td></td></tr><tr><td>Cardamonin</td><td>Tocris or equivalent</td><td>2509</td><td>Anti-inflammatory, used as positive control</td></tr><tr><td>SKF 86002&amp;nbsp;</td><td>Tocris or equivalent</td><td>2008</td><td>Anti-inflammatory, used as positive control</td></tr><tr><td>DMSO</td><td>Sigma or equivalent</td><td>D8418</td><td></td></tr><tr><td>48% hydrofluoric acid</td><td>Sigma or equivalent</td><td>339261</td><td>Corrosive and acute toxicity</td></tr><tr><td>1000 &amp;mu;L Single channel pipettors</td><td>Rainin or equivalent</td><td>17014382</td><td></td></tr><tr><td>200 &amp;mu;L Single channel pipettors</td><td>Rainin or equivalent</td><td>17014391</td><td></td></tr><tr><td>20 &amp;mu;L Single channel pipettors</td><td>Rainin or equivalent</td><td>17014392</td><td></td></tr><tr><td>1000 &amp;mu;L 12-channel pipettors</td><td>Rainin or equivalent</td><td>17014497</td><td></td></tr><tr><td>200 &amp;mu;L 12-channel pipettors</td><td>Rainin or equivalent</td><td>17013810</td><td></td></tr><tr><td>20 &amp;mu;L 12-channel pipettors</td><td>Rainin or equivalent</td><td>17013808</td><td></td></tr><tr><td>Pipettor tips 1000 &amp;mu;L</td><td>Rainin or equivalent</td><td>17002920</td><td></td></tr><tr><td>Pipettor tips 200 &amp;mu;L</td><td>Rainin or equivalent</td><td>17014294</td><td></td></tr><tr><td>Pipettor tips 20 &amp;mu;L</td><td>Rainin or equivalent</td><td>17002928</td><td></td></tr><tr><td>Chemical fume hood</td><td>Jamestown Metal Products</td><td>MHCO_229</td><td></td></tr><tr><td>384-well sample storage plates</td><td>Thermo or equivalent</td><td>262261</td><td></td></tr><tr><td>Sodium chloride</td><td>Sigma or equivalent</td><td>S6191</td><td></td></tr><tr><td>50 mL conical tubes</td><td>Thermo or equivalent</td><td>14-959-49A</td><td></td></tr><tr><td>Serological pipettes 50 mL</td><td>Corning or equivalent</td><td>07-200-576</td><td></td></tr><tr><td>Serological pipettes 25 mL</td><td>Corning or equivalent</td><td>07-200-575</td><td></td></tr><tr><td>Serological pipettes 10 mL</td><td>Corning or equivalent</td><td>07-200-574</td><td></td></tr><tr><td>Serological pipettes 5 mL</td><td>Corning or equivalent</td><td>07-200-573</td><td></td></tr><tr><td>SV40-HCEC immortalized human corneal epithelial cells</td><td>N/A</td><td>N/A</td><td>These cells are not commercially available, but can be obtained from the investigators cited in the article</td></tr><tr><td>Sceptor Handheld Automated Cell Counter</td><td>Millipore or equivalent</td><td>PHCC20060</td><td></td></tr><tr><td>GeneTitan Multi-Channel (MC) Instrument</td><td>Affymetrix or equivalent</td><td>00-0372</td><td></td></tr><tr><td>Affymetrix 24- and 96-array plates</td><td>Affymetrix or equivalent</td><td>901257; 901434</td><td></td></tr><tr><td>Draegger tube HF</td><td>Draeger or equivalent</td><td>8103251</td><td></td></tr><tr><td>Draegger tube CP</td><td>Draeger or equivalent</td><td>8103421</td><td></td></tr><tr><td>Draegger pump</td><td>Draeger or equivalent</td><td>6400000</td><td></td></tr><tr><td>Clear Plate seals</td><td>Resesarch Products International or Equivalent</td><td>202502</td><td></td></tr><tr><td>Reagent reservoirs</td><td>VistaLab Technologies or equivalent</td><td>3054-1000</td><td></td></tr><tr><td>Xlfit</td><td>IDBS or equivalent</td><td>N/A</td><td>Excel add-in used for automated curve fitting</td></tr></tbody></table>

high throughput sirna screening for chloropicrin and hydrogen fluoride-induced cornea epithelial cell injury

Toxicant-induced ocular injury is a true ocular emergency because chemicals have the potential to rapidly inflict significant tissue damage. Treatments for toxicant-induced corneal injury are generally supportive as no specific therapeutics exist to treat these injuries. In the efforts to develop treatments and therapeutics to care for exposure, it can be important to understand the molecular and cellular mechanisms of these injuries. We propose that utilization of high throughput small inhibitory RNA (siRNA) screening can be an important tool that could help to more rapidly elucidate the molecular mechanisms of chemical cornea epithelial injury. siRNA are double stranded RNA molecules that are 19-25 nucleotides long and utilize the post-transcriptional gene silencing pathway to degrade mRNA which have homology to the siRNA. The resulting reduction of expression of the specific gene can then be studied in toxicant exposed cells to ascertain the function of that gene in the cellular response to the toxicant. The development and validation of in vitro exposure models and methods for the high throughput screening (HTS) of hydrogen fluoride- (HF) and chloropicrin- (CP) induced ocular injury are presented in this article. Although we selected these two toxicants, our methods are applicable to the study of other toxicants with minor modifications to the toxicant exposure protocol. The SV40 large T antigen immortalized human corneal epithelial cell line SV40-HCEC was selected for study. Cell viability and IL-8 production were selected as endpoints in the screening protocol. Several challenges associated with the development of toxicant exposure and cell culture methods suitable for HTS studies are presented. The establishment of HTS models for these toxicants allows for further studies to better understand the mechanism of injury and to screen for potential therapeutics for chemical ocular injury.

Exposure Method Development
We refined and evaluated the suitability of the human corneal epithelial cell line SV40-HCEC for use in HTS studies. SV40-HCEC were immortalized using the SV-40 large T antigen and were a gift from Dhanajay Pal23. There were too many variables explored in exposure methodology development to present concisely herein, and so, only some samples of findings we beli...

Watch this Scientific Journal Video about High Throughput SiRNA Screening for Chloropicrin and Hydrogen Fluoride-Induced Cornea Epithelial Cell Injury at JoVE.com

High Throughput SiRNA Screening for Chloropicrin and Hydrogen Fluoride-Induced Cornea Epithelial Cell Injury

High throughput small inhibitory RNA screening is an important tool that could help to more rapidly elucidate the molecular mechanisms of chemical cornea epithelial injury. Herein, we present the development and validation of exposure models and methods for the high throughput screening of hydrogen fluoride- and chloropicrin-induced cornea epithelial injury.

Toxicant-induced ocular injury is a true ocular emergency because chemicals have the potential to rapidly inflict significant tissue damage. Treatments ...

high-throughput-sirna-screening-for-chloropicrin-hydrogen-fluoride

Research

JoVE Journal

Biology

5.8K Views.  US Army Medical Research Institute of Chemical Defense. Hoge-doorvoer kleine remmende RNA screening is een belangrijk instrument die helpen kan sneller het ophelderen van de moleculaire mechanismen van chemische hoornvlies epitheliale letsel. Hierin presenteren wij de ontwikkeling en validatie van blootstelling modellen en methoden mbt de hoge doorvoer screening van waterstoffluoride en chloorpikrine-geïnduceerde hoornvlies epitheliale letsel.

Video: High Throughput SiRNA Screening for Chloropicrin and Hydrogen Fluoride-Induced Cornea Epithelial Cell Injury

RNA Interference-based Investigation of the Function of Heat Shock Protein 27 during Corneal Epithelial Wound Healing

Small interfering RNA (siRNA) is among the most widely used RNA interference methods for the short-term silencing of protein-coding genes. siRNA is a synthetic RNA duplex created to specifically target a mRNA transcript to induce its degradation and it has been used to identify novel pathways in various cellular processes. Few reports exist regarding the role of phosphorylated heat shock protein 27 (HSP27) in corneal epithelial wound healing. Herein, cultured human corneal epithelial cells were divided into a scrambled control-siRNA transfected group and a HSP27-specific siRNA-transfected group. Scratch-induced directional wounding assays, and western blotting, and flow cytometry were then performed. We conclude that HSP27 has roles in corneal epithelial wound healing that may involve epithelial cell apoptosis and migration. Here, step-by-step descriptions of sample preparation and the study protocol are provided.

Herein, we present a protocol to use heat shock protein 27 (HSP27)-specific small interfering RNA to assess the function of HSP27 during corneal epithelial wound healing. RNA interference is the best method for effectively knocking-down gene expression to investigate protein function in various cell types.

Small interfering RNA (siRNA) is among the most widely used RNA interference methods for the short-term silencing of protein-coding genes. siRNA is a ...

Genetics

Drug-induced Sensitization of Adenylyl Cyclase: Assay Streamlining and Miniaturization for Small Molecule and siRNA Screening Applications

Sensitization of adenylyl cyclase (AC) signaling has been implicated in a variety of neuropsychiatric and neurologic disorders including substance abuse and Parkinson&#39;s disease. Acute activation of G&#945;i/o-linked receptors inhibits AC activity, whereas persistent activation of these receptors results in heterologous sensitization of AC and increased levels of intracellular cAMP. Previous studies have demonstrated that this enhancement of AC responsiveness is observed both in vitro and in vivo following the chronic activation of several types of G&#945;i/o-linked receptors including D2 dopamine and &#956; opioid receptors. Although heterologous sensitization of AC was first reported four decades ago, the mechanism(s) that underlie this phenomenon remain largely unknown. The lack of mechanistic data presumably reflects the complexity involved with this adaptive response, suggesting that nonbiased approaches could aid in identifying the molecular pathways involved in heterologous sensitization of AC. Previous studies have implicated kinase and Gb&#947; signaling as overlapping components that regulate the heterologous sensitization of AC. To identify unique and additional overlapping targets associated with sensitization of AC, the development and validation of a scalable cAMP sensitization assay is required for greater throughput. Previous approaches to study sensitization are generally cumbersome involving continuous cell culture maintenance as well as a complex methodology for measuring cAMP accumulation that involves multiple wash steps. Thus, the development of a robust cell-based assay that can be used for high throughput screening (HTS) in a 384&#160;well format would facilitate future studies. Using two D2 dopamine receptor cellular models (i.e.&#160;CHO-D2L and HEK-AC6/D2L), we have converted our 48-well sensitization assay (&#62;20 steps 4-5 days) to a five-step, single day assay in 384-well format. This new format is amenable to small molecule screening, and we demonstrate that this assay design can also be readily used for reverse transfection of siRNA in anticipation of targeted siRNA library screening.

Persistent activation of inhibitory G protein-coupled receptors results in sensitization of adenylyl cyclase signaling. To identify the essential molecular pathways, nonbiased approaches are necessary; however, this strategy requires the development of a scalable cell-based cAMP sensitization assay. Herein, we describe a sensitization assay for small molecule and siRNA screening.

Sensitization of adenylyl cyclase (AC) signaling has been implicated in a variety of neuropsychiatric and neurologic disorders including substance abuse ...

Bioengineering

High-throughput Screening for Broad-spectrum Chemical Inhibitors of RNA Viruses

RNA viruses are responsible for major human diseases such as flu, bronchitis, dengue, Hepatitis C or measles. They also represent an emerging threat because of increased worldwide exchanges and human populations penetrating more and more natural ecosystems. A good example of such an emerging situation is chikungunya virus epidemics of 2005-2006 in the Indian Ocean. Recent progresses in our understanding of cellular pathways controlling viral replication suggest that compounds targeting host cell functions, rather than the virus itself, could inhibit a large panel of RNA viruses. Some broad-spectrum antiviral compounds have been identified with host target-oriented assays. However, measuring the inhibition of viral replication in cell cultures using reduction of cytopathic effects as a readout still represents a paramount screening strategy. Such functional screens have been greatly improved by the development of recombinant viruses expressing reporter enzymes capable of bioluminescence such as luciferase. In the present report, we detail a high-throughput screening pipeline, which combines recombinant measles and chikungunya viruses with cellular viability assays, to identify compounds with a broad-spectrum antiviral profile.

In vitro assays to measure virus replication have been greatly improved by the development of recombinant RNA viruses expressing luciferase or other enzymes capable of bioluminescence. Here we detail a high-throughput screening pipeline that combines such recombinant strains of measles and chikungunya viruses to isolate broad-spectrum antivirals from chemical libraries.

RNA viruses are responsible for major human diseases such as flu, bronchitis, dengue, Hepatitis C or measles. They also represent an emerging threat ...

Immunology and Infection

Assessment of Oxidative Damage in the Primary Mouse Ocular Surface Cells/Stem Cells in Response to Ultraviolet-C (UV-C) Damage

The ocular surface is subjected to regular wear and tear due to various environmental factors. Exposure to UV-C radiation constitutes an occupational health hazard. Here, we demonstrate the exposure of primary stem cells from the mouse ocular surface to UV-C radiation. Reactive oxygen species (ROS) formation is the readout of the extent of oxidative stress/damage. In an experimental in vitro setting, it is also essential to assess the percentage of dead cells generated due to oxidative stress. In this article, we will demonstrate the 2&#39;,7&#39;-Dichlorofluoresceindiacetate (DCFDA) staining of UV-C exposed mouse primary ocular surface stem cells and their quantification based on the fluorescent images of DCFDA staining. DCFDA staining directly corresponds to ROS generation. We also demonstrate the quantification of dead and live cells by simultaneous staining with propidium iodide (PI) and Hoechst 3332 respectively and the percentage of DCFDA (ROS positive) and PI positive cells.

Assessment of Oxidative Damage in the Primary Mouse Ocular Surface Cells/Stem Cells in Response to Ultraviolet-C UV-C Damage

This protocol demonstrates the simultaneous detection of reactive oxygen species (ROS), live cells, and dead cells in live primary cultures from mouse ocular surface cells. 2&#39;,7&#39;-Dichlorofluoresceindiacetate, propidium iodide, and Hoechst staining are used to assess the ROS, dead cells, and live cells, respectively, followed by imaging and analysis.

The ocular surface is subjected to regular wear and tear due to various environmental factors. Exposure to UV-C radiation constitutes an occupational ...

Developmental Biology

Toxicity Screens in Human Retinal Organoids for Pharmaceutical Discovery

Organoids provide a promising platform to study disease mechanism and treatments, directly in the context of human tissue with the versatility and throughput of cell culture. Mature human retinal organoids are utilized to screen potential pharmaceutical treatments for the age-related retinal degenerative disease macular telangiectasia type 2 (MacTel).
We have recently shown that MacTel can be caused by elevated levels of an atypical lipid species, deoxysphingolipids (deoxySLs). These lipids are toxic to the retina and may drive the photoreceptor loss that occurs in MacTel patients. To screen drugs for their ability to prevent deoxySL photoreceptor toxicity, we generated human retinal organoids from a non-MacTel induced pluripotent stem cell (iPSC) line and matured them to a post-mitotic age where they develop all of the neuronal lineage-derived cells of the retina, including functionally mature photoreceptors. The retinal organoids were treated with a deoxySL metabolite and apoptosis was measured within the photoreceptor layer using immunohistochemistry. Using this toxicity model, pharmacological compounds that prevent deoxySL-induced photoreceptor death were screened. Using a targeted candidate approach, we determined that fenofibrate, a drug commonly prescribed for the treatment of high cholesterol and triglycerides, can also prevent deoxySL toxicity in the cells of the retina.
The toxicity screen successfully identified an FDA-approved drug that can prevent photoreceptor death. This is a directly actionable finding owing to the highly disease-relevant model tested. This platform can be easily modified to test any number of metabolic stressors and potential pharmacological interventions for future treatment discovery in retinal diseases.

Here we present a step-by-step protocol to generate mature human retinal organoids and utilize them in a photoreceptor toxicity assay to identify pharmaceutical candidates for the age-related retinal degenerative disease macular telangiectasia type 2 (MacTel).

Organoids provide a promising platform to study disease mechanism and treatments, directly in the context of human tissue with the versatility and ...

Novel ROS Quantification Using Dihydroethidium in Liver Cancer Cells

High Throughput Screening Assessment of Reactive Oxygen Species (ROS) Generation using Dihydroethidium (DHE) Fluorescence Dye

Reactive oxygen species (ROS) play a key role in the regulation of cellular metabolism in physiological and pathological processes. Physiological ROS production plays a central role in the spatial and temporal modulation of normal cellular functions such as proliferation, signaling, apoptosis, and senescence. In contrast, chronic ROS overproduction is responsible for a wide spectrum of diseases, such as cancer, cardiovascular disease, and diabetes, among others. Quantifying ROS levels in an accurate and reproducible manner is thus essential to understanding normal cellular functionality. Fluorescence imaging-based methods to characterize intra-cellular ROS species is a common approach. Many of the imaging ROS protocols in the literature use 2&#39;-7&#39;-dichlorodihydrofluorescein diacetate (DCFH-DA) dye. However, this dye suffers from significant limitations in its usage and interpretability. The current protocol demonstrates the use of a dihydroethidium (DHE) fluorescent probe as an alternative method to quantify total ROS production in a high-throughput setting. The high throughput imaging platform, CX7 Cellomics, was used to measure and quantify the ROS production. This study was conducted in three hepatocellular cancer cell lines - HepG2, JHH4, and HUH-7. This protocol provides an in-depth description of the various procedures involved in the assessment of ROS within the cells, including - preparation of DHE solution, incubation of cells with DHE solution, and measurement of DHE intensity necessary to characterize the ROS production. This protocol demonstrates that DHE fluorescent dye is a robust and reproducible choice to characterize intracellular ROS production in a high-throughput manner. High throughput approaches to measure ROS production are likely to be helpful in a variety of studies, such as toxicology, drug screening, and cancer biology.

Author Spotlight: High-Throughput Measurement of Intracellular ROS Levels in Hepatocellular Lines

This protocol describes a novel method to quantify intracellular reactive oxygen species (ROS) using dihydroethidium (DHE) as a fluorescence dye probe using a high-throughput screening approach. The protocol describes the methods for quantitative assessment of intracellular reactive oxygen species (ROS) in the three different hepatocellular carcinoma cell lines.

Reactive oxygen species (ROS) play a key role in the regulation of cellular metabolism in physiological and pathological processes. Physiological ROS ...

siRNA Screening to Identify Ubiquitin and Ubiquitin-like System Regulators of Biological Pathways in Cultured Mammalian Cells

Post-translational modification of proteins with ubiquitin and ubiquitin-like molecules (UBLs) is emerging as a dynamic cellular signaling network that regulates diverse biological pathways including the hypoxia response, proteostasis, the DNA damage response and transcription.&#160; To better understand how UBLs regulate pathways relevant to human disease, we have compiled a human siRNA &#8220;ubiquitome&#8221; library consisting of 1,186 siRNA duplex pools targeting all known and predicted components of UBL system pathways. This library can be screened against a range of cell lines expressing reporters of diverse biological pathways to determine which UBL components act as positive or negative regulators of the pathway in question.&#160; Here, we describe a protocol utilizing this library to identify ubiquitome-regulators of the HIF1A-mediated cellular response to hypoxia using a transcription-based luciferase reporter.&#160; An initial assay development stage is performed to establish suitable screening parameters of the cell line before performing the screen in three stages: primary, secondary and tertiary/deconvolution screening. &#160;The use of targeted over whole genome siRNA libraries is becoming increasingly popular as it offers the advantage of reporting only on members of the pathway with which the investigators are most interested.&#160; Despite inherent limitations of siRNA screening, in particular false-positives caused by siRNA off-target effects, the identification of genuine novel regulators of the pathways in question outweigh these shortcomings, which can be overcome by performing a series of carefully undertaken control experiments.

Here, we describe a methodology to perform a targeted siRNA &#8220;ubiquitome&#8221; screen to identify novel ubiquitin and ubiquitin-like regulators of the HIF1A-mediated cellular response to hypoxia.&#160; This can be adapted to any biological pathway where a robust read out of reporter activity is available.

Post-translational modification of proteins with ubiquitin and ubiquitin-like molecules (UBLs) is emerging as a dynamic cellular signaling network that ...

High-throughput Screening for Chemical Modulators of Post-transcriptionally Regulated Genes

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays focus on transcriptional regulation, being essentially aimed at the identification of promoter-targeting molecules. As a result, post-transcriptional mechanisms are largely uncovered by gene expression targeted drug development. Here we describe a cell-based assay aimed at investigating the role of the 3&#39; untranslated region (3&#8217; UTR) in the modulation of the fate of its mRNA, and at identifying compounds able to modify it. The assay is based on the use of a luciferase reporter construct containing the 3&#8217; UTR of a gene of interest stably integrated into a disease-relevant cell line. The protocol is divided into two parts, with the initial focus on the primary screening aimed at the identification of molecules affecting luciferase activity after 24 hr of treatment. The second part of the protocol describes the counter-screening necessary to discriminate compounds modulating luciferase activity specifically through the 3&#8217; UTR. In addition to the detailed protocol and representative results, we provide important considerations about the assay development and the validation of the hit(s) on the endogenous target. The described cell-based reporter gene assay will allow scientists to identify molecules modulating protein levels via post-transcriptional mechanisms dependent on a 3&#8217; UTR.

Here we describe a cell-based reporter gene assay as a valuable tool to screen chemical libraries for compounds modulating post-transcriptional control mechanisms exerted through 3&#8217; UTR.

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays ...

Determining the Toxicity of UV Radiation and Chemicals on Primary and Immortalized Human Corneal Epithelial Cells

This article describes the methods of measuring the toxicity of ultraviolet (UV) radiation and ocular toxins on primary (pHCEC) and immortalized (iHCEC) human corneal epithelial cell cultures. Cells were exposed to UV radiation and toxic doses of benzalkonium chloride (BAK), hydrogen peroxide (H2O2), and sodium dodecyl sulfate (SDS). Metabolic activity was measured using a metabolic assay. The release of inflammatory cytokines was measured using a multi-plex interleukin (IL)-1&#946;, IL-6, IL-8, and tumor necrosis factor-alpha (TNF-&#945;) assay, and cells were evaluated for viability using fluorescent dyes. 
The damaging effects of UV on cell metabolic activity and cytokine release occurred at 5 min of UV exposure for iHCEC and 20 min for pHCEC. Similar percent drops in metabolic activity of the iHCEC and pHCEC occurred after exposure to BAK, H2O2, or SDS, and the most significant changes in cytokine release occurred for IL-6 and IL-8. Microscopy of fluorescently stained iHCEC and pHCEC BAK-exposed cells showed cell death at 0.005% BAK exposure, although the degree of ethidium staining was greater in the iHCECs than pHCECs. Utilizing multiple methods of assessing toxic effects using microscopy, assessments of metabolic activity, and cytokine production, the toxicity of UV radiation and chemical toxins could be determined for both primary and immortalized cell lines.

This article describes the procedures used to evaluate the toxicity of UV radiation and chemical toxins on a primary and immortalized cell line.

This article describes the methods of measuring the toxicity of ultraviolet (UV) radiation and ocular toxins on primary (pHCEC) and immortalized (iHCEC) ...

Ex Vivo Porcine Corneal Culture System for Toxicity Testing and Wound Healing

Evaluating Therapeutic and Chemical Toxicity Using Organ-Cultured Porcine Corneas and Epithelial Wound Healing

Due to its anatomical and physiological similarities to the human eye, the porcine eye serves as a robust model for biomedical research and ocular toxicity assessment. An air/liquid corneal culture system using porcine eyes was developed, and ex vivo epithelial wound healing was utilized as a critical parameter for these studies. Fresh pig corneas were processed for organ culture, with or without epithelial wounding. The corneas were cultured in a humidified 5% CO2 incubator at 37 &#176;C in MEM, with or without testing agents. Corneal permeability and wound healing rates were measured, and epithelial cells and/or whole corneas can be processed for immunohistochemistry, western blotting, and qPCR for molecular and cellular analyses. This study describes a detailed protocol and presents two studies using this ex vivo system. The data show that porcine corneal organ culture, combined with epithelial wound healing, is a suitable ex vivo model for chemical toxicity testing, studying diabetic keratopathy, and identifying potential therapies.

Author Spotlight: Exploring Corneal Innate Immunity and Delayed Wound Healing in Diabetic Patients

Porcine corneal ex vivo organ culture and epithelial wound healing provide an economical, ethical, reproducible, and quantitative means for testing the ocular toxicity of chemicals. They also aid in elucidating mechanisms underlying the regulation of epithelialization and tissue repair, and in evaluating therapeutics for treating diabetic keratopathy and delayed wound healing.

High Throughput SiRNA Screening for Chloropicrin and Hydrogen Fluoride-Induced Cornea Epithelial Cell Injury

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