Name: high throughput sirna screening for chloropicrin and hydrogen fluoride-induced cornea epithelial cell injury
Uploaded: 2018-06-16T13:00:00
Description: Watch this Scientific Journal Video about High Throughput SiRNA Screening for Chloropicrin and Hydrogen Fluoride-Induced Cornea Epithelial Cell Injury at JoVE.com

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.

1. Cell Culture Maintenance
<ol>
	<li>Grow the cell line SV40-HCEC at 37 &#176;C, 5% CO2, and 90% humidity in DMEM F-12 with 15% fetal bovine serum (FBS), 1% L-glutamine, 10 &#181;g/L epidermal growth factor (EGF), and 5 mg/L insulin.</li>
	<li>Passage the cell line every 3 to 4 days (depending on seeding density) to ensure that confluency never exceeds 80% during culture maintenance.</li>
	<li>Detach the cells from flasks using a detachment solution (14 mL solution for every 150 cm2 flask) and in...

<ol>
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N., Shepler, K., Lawrence, L. J., Lee, H., Seiber, J. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Fumigants: Environmental Fate, Exposure, and Analysis. 652, (1997).</li><li>Adamek, E., Pawlowska-Goral, K., Bober, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+and+in+vivo+effects+of+fluoride+ions+on+enzyme+activity.">In vitro and in vivo effects of fluoride ions on enzyme activity.</a> Annales Academiae Medicae Stetinensis. 51 (2), 69-85 (2005).</li><li>Heard, K., Hill, R. E., Cairns, C. B., Dart, R. 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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.

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			<td height="21" style="height:21px;width:347px;">Bravo liquid handing platform</td>
			<td style="width:247px;">Agilent or equivalent</td>
			<td style="width:119px;">G5409A</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">Bravo plate shaker</td>
			<td style="width:247px;">Agilent or equivalent</td>
			<td style="width:119px;">Option 159</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">Bravo 96LT disposable tip head</td>
			<td style="width:247px;">Agilent or equivalent</td>
			<td style="width:119px;">Option 178</td>
			<td>96-channel large tip pipetting head unit</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">Bravo 96ST disposable tip head</td>
			<td style="width:247px;">Agilent or equivalent</td>
			<td style="width:119px;">Option 177</td>
			<td>96-channel small tip pipetting head unit</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">Bravo 384ST disposable tip head</td>
			<td style="width:247px;">Agilent or equivalent</td>
			<td style="width:119px;">Option 179</td>
			<td>384-channel small tip pipetting head unit</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">Bravo 96 250 &#956;L sterile barrier tips</td>
			<td style="width:247px;">Agilent or equivalent</td>
			<td style="width:119px;">19477-022</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">Bravo 384 30 &#956;L sterile barrier tips</td>
			<td style="width:247px;">Agilent or equivalent</td>
			<td style="width:119px;">19133-212</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">Bravo 384 70 &#956;L sterile barrier tips</td>
			<td style="width:247px;">Agilent or equivalent</td>
			<td style="width:119px;">19133-212</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">EnSpire multimode plate reader</td>
			<td style="width:247px;">Perkin Elmer or equivalent</td>
			<td style="width:119px;">2300-0000</td>
			<td>AlphaLISA assay detector with high power laser excitation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">IL-8 (human) AlphaLISA Detection Kit&#160;</td>
			<td style="width:247px;">Perkin Elmer or equivalent</td>
			<td style="width:119px;">AL224F</td>
			<td>no-wash bead-based assay</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">ProxiPlate-384 Plus white 384-shallow well microplates</td>
			<td style="width:247px;">Perkin Elmer or equivalent</td>
			<td>6008359</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">Lipofectamine RNAiMAX</td>
			<td style="width:247px;">Invitrogen or equivalent</td>
			<td style="width:119px;">13778500</td>
			<td>Transfection reagent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">Opti-MEM 1 Reduced Serum Medium</td>
			<td style="width:247px;">Invitrogen or equivalent</td>
			<td style="width:119px;">31985070</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">TrypLE Express</td>
			<td style="width:247px;">Gibco or equivalent</td>
			<td style="width:119px;">12605010</td>
			<td>Cell detachment solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">IncuCyte Zoom</td>
			<td style="width:247px;">Essen Instruments or equivalent</td>
			<td style="width:119px;">ESSEN BIOSCI 4473</td>
			<td>Incubator-housed automated microscope</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">Chloropicrin</td>
			<td style="width:247px;">Trinity Manufacturing or equivalent</td>
			<td style="width:119px;">N/A</td>
			<td>Acute toxicity and irritant</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">DMEM-F12 cell culture medium</td>
			<td style="width:247px;">Invitrogen or equivalent</td>
			<td>11330-057</td>
			<td>Contains HEPES</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">Fetal bovine serum</td>
			<td style="width:247px;">Invitrogen or equivalent</td>
			<td>1891471</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">Human epidermal growth factor (cell culture grade)</td>
			<td style="width:247px;">Invitrogen or equivalent</td>
			<td>E9644-.2MG</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">Recombinant human insulin (cell culture grade)</td>
			<td style="width:247px;">Invitrogen or equivalent</td>
			<td>12585-014</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">Penicillin-Streptomycin solution (cell culture grade)</td>
			<td style="width:247px;">Invitrogen or equivalent</td>
			<td>15140122</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">Hydrocortisone (cell culture grade)</td>
			<td style="width:247px;">Sigma or equivalent</td>
			<td>H0888-10G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">Glucose&#160; (cell culture grade)</td>
			<td style="width:247px;">Sigma or equivalent</td>
			<td style="width:119px;">G7021</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">PBS&#160; (cell culture grade)</td>
			<td style="width:247px;">Sigma or equivalent</td>
			<td style="width:119px;">P5493</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">siRNA</td>
			<td style="width:247px;">Dharmacon or equivalent</td>
			<td style="width:119px;">various</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Thiazolyl blue tetrazolium bromide</td>
			<td style="width:247px;">Sigma or equivalent</td>
			<td style="width:119px;">M5655</td>
			<td>MTT assay substrate</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">siRNA buffer</td>
			<td style="width:247px;">Thermo or equivalent</td>
			<td style="width:119px;">B002000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">96-well cell culture plates</td>
			<td style="width:247px;">Corning or equivalent</td>
			<td style="width:119px;">CLS3595</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">T150 cell culture flasks</td>
			<td style="width:247px;">Corning or equivalent</td>
			<td style="width:119px;">CLS430825</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">BSL-2 cell culture hood</td>
			<td style="width:247px;">Nuaire or equivalent</td>
			<td style="width:119px;">NU-540</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">300 mL robotic reservoirs</td>
			<td style="width:247px;">Thermo or equivalent</td>
			<td style="width:119px;">12-565-572&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">96 baffled automation reservoirs</td>
			<td style="width:247px;">Thermo or equivalent</td>
			<td style="width:119px;">1064-15-8</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">500 mL sterile disposable storage bottles</td>
			<td style="width:247px;">Corning or equivalent</td>
			<td style="width:119px;">CLS430282</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">Microplate heat sealer</td>
			<td style="width:247px;">Thermo or equivalent</td>
			<td style="width:119px;">AB-1443A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">Microplate heat sealing foil</td>
			<td style="width:247px;">Thermo or equivalent</td>
			<td style="width:119px;">AB-0475</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">Cardamonin</td>
			<td style="width:247px;">Tocris or equivalent</td>
			<td style="width:119px;">2509</td>
			<td>Anti-inflammatory, used as positive control</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">SKF 86002&#160;</td>
			<td style="width:247px;">Tocris or equivalent</td>
			<td style="width:119px;">2008</td>
			<td>Anti-inflammatory, used as positive control</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">DMSO</td>
			<td style="width:247px;">Sigma or equivalent</td>
			<td style="width:119px;">D8418</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">48% hydrofluoric acid</td>
			<td style="width:247px;">Sigma or equivalent</td>
			<td style="width:119px;">339261</td>
			<td>Corrosive and acute toxicity</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">1000 &#956;L Single channel pipettors</td>
			<td style="width:247px;">Rainin or equivalent</td>
			<td style="width:119px;">17014382</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">200 &#956;L Single channel pipettors</td>
			<td style="width:247px;">Rainin or equivalent</td>
			<td style="width:119px;">17014391</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">20 &#956;L Single channel pipettors</td>
			<td style="width:247px;">Rainin or equivalent</td>
			<td style="width:119px;">17014392</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">1000 &#956;L 12-channel pipettors</td>
			<td style="width:247px;">Rainin or equivalent</td>
			<td style="width:119px;">17014497</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">200 &#956;L 12-channel pipettors</td>
			<td style="width:247px;">Rainin or equivalent</td>
			<td style="width:119px;">17013810</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">20 &#956;L 12-channel pipettors</td>
			<td style="width:247px;">Rainin or equivalent</td>
			<td style="width:119px;">17013808</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">Pipettor tips 1000 &#956;L</td>
			<td style="width:247px;">Rainin or equivalent</td>
			<td style="width:119px;">17002920</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">Pipettor tips 200 &#956;L</td>
			<td style="width:247px;">Rainin or equivalent</td>
			<td style="width:119px;">17014294</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">Pipettor tips 20 &#956;L</td>
			<td style="width:247px;">Rainin or equivalent</td>
			<td style="width:119px;">17002928</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">Chemical fume hood</td>
			<td style="width:247px;">Jamestown Metal Products</td>
			<td style="width:119px;">MHCO_229</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">384-well sample storage plates</td>
			<td style="width:247px;">Thermo or equivalent</td>
			<td style="width:119px;">262261</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">Sodium chloride</td>
			<td style="width:247px;">Sigma or equivalent</td>
			<td style="width:119px;">S6191</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">50 mL conical tubes</td>
			<td style="width:247px;">Thermo or equivalent</td>
			<td style="width:119px;">14-959-49A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">Serological pipettes 50 mL</td>
			<td style="width:247px;">Corning or equivalent</td>
			<td style="width:119px;">07-200-576</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">Serological pipettes 25 mL</td>
			<td style="width:247px;">Corning or equivalent</td>
			<td style="width:119px;">07-200-575</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">Serological pipettes 10 mL</td>
			<td style="width:247px;">Corning or equivalent</td>
			<td style="width:119px;">07-200-574</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">Serological pipettes 5 mL</td>
			<td style="width:247px;">Corning or equivalent</td>
			<td style="width:119px;">07-200-573</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:347px;">SV40-HCEC immortalized human corneal epithelial cells</td>
			<td style="width:247px;">N/A</td>
			<td style="width:119px;">N/A</td>
			<td>These cells are not commercially available, but can be obtained from the investigators cited in the article</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">Sceptor Handheld Automated Cell Counter</td>
			<td style="width:247px;">Millipore or equivalent</td>
			<td style="width:119px;">PHCC20060</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">GeneTitan Multi-Channel (MC) Instrument</td>
			<td style="width:247px;">Affymetrix or equivalent</td>
			<td style="width:119px;">00-0372</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">Affymetrix 24- and 96-array plates</td>
			<td style="width:247px;">Affymetrix or equivalent</td>
			<td style="width:119px;">901257; 901434</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">Draegger tube HF</td>
			<td style="width:247px;">Draeger or equivalent</td>
			<td style="width:119px;">8103251</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">Draegger tube CP</td>
			<td style="width:247px;">Draeger or equivalent</td>
			<td style="width:119px;">8103421</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">Draegger pump</td>
			<td style="width:247px;">Draeger or equivalent</td>
			<td style="width:119px;">6400000</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:347px;">Clear Plate seals</td>
			<td style="width:247px;">Resesarch Products International or Equivalent</td>
			<td style="width:119px;">202502</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:347px;">Reagent reservoirs</td>
			<td style="width:247px;">VistaLab Technologies or equivalent</td>
			<td style="width:119px;">3054-1000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">Xlfit</td>
			<td style="width:247px;">IDBS or equivalent</td>
			<td style="width:119px;">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 ...

This video shows our methods for high throughput siRNA screening in a corneal epithelial cell model of toxicant injury. This work is important, because high throughput siRNA screening is a cost-efficient and time-efficient method to elucidate pathways and regulators of the cellular response to toxicants. This work may help further our understanding of how the cornea responds to toxicant insults.Prepare a suspension of cornea epithelial cells in pre-warmed medium containing 0.5 micrograms per milliliter Hydrocortisone in a disposable medium bottle. Swirl the bottle to evenly suspend the cells, and pour into a reservoir on the orbital shaker nest of an automated liquid handler. Keep the cell suspension on a 37 degree warming plate during the cell plating process.Use an automated liquid handler to add cells to plates at a density of one thousand cells per well in 70 microliters of medium per well on a 96-well plate. Seed a minimum of 14 96-well plates with cells for every siRNA library plate to be studied. Run the orbital shaker at 100 cycles per minute constantly during the seeding process, and use a 50 microliter per second pipetting speed for all steps.Mix the cell suspension using the automated liquid handler before seeding the first plate. Aspirate 140 microliters of the cell suspension, and dispense 70 microliters into each well of two cell culture plates. Repeat the mixing and dispensing until all plates have been seeded with cells.Refill the cell suspension reservoir as needed. After the plates have been seeded, remove them from the liquid handler and incubate them for 30 minutes at room temperature. Then, transfer them to a cell culture incubator.Evaluate the cell density of all plates the following morning using an automated imaging system, which is housed in the cell culture incubator. Exclude plates from study which have interior wells that are not between 15 percent and 22 percent confluent. Transfect the cells 24 hours after seeding w ith four picomoles per well of siRNA and 0.3 microliters per well of transfection reagent, as follows.Acquire siRNA library plates from the vendor, configured to contain 80 siRNA targets per plate with columns one and twelve left empty. Reconstitute the siRNA library plate with siRNA buffer according to the manufacturer's instructions to a final concentration of two picomoles per microliter for each well of siRNA. Perform all the transfections using an automated liquid handler and a 25 microliter per second pipetting speed for all steps.Use pre-configured tip boxes to address only the wells that will be transfected. Use the top half of the library plate to transfect a set of six replicate plates, referred to as the Top Plate Set"Use the bottom half of the library plate to transfect a different set of six replica plates, referred to as the Bottom Plate Set"Together, the twelve plates comprise a full plate set, and each cell plate contains the same set of controls. Transfect wells B2 to B6 of all plates with negative pool siRNA after all top and bottom plate sets have been transfected with library siRNA.Also transfect wells B2 to B6 of another two plates that do not receive library siRNA with negative pool siRNA. These will serve as unexposed controls. Incubate all cell plates in a cell culture incubator for four hours after transfection mixes have been added, and mix by gentle tapping every hour.Use an automated liquid handler to wash the plates twice with pre-warmed medium, diluted one to five in PBS and re-fed with 100 microliters per well pre-warmed medium. Use a 50 microliter per second pipetting speed for all steps. Use wells C2 to C6 as non-transfected controls.Keep wells B7 to B11 and C7 to C11 non-transfected, to be used as drug and vehicle controls for toxicant exposure. 24 hours after transfection, use an automated liquid handler to re-feed the plates. Use a 50 microliter per second pipetting speed for all steps.Aspirate 100 microliters from each well, and discard that into a waste reservoir. Re-feed each well with 100 microliters per well pre-warmed H-Cort.medium. Empty the waste reservoir as needed.Refill the reservoir with medium as needed. Two days after transfection and two hours prior to exposure, prepare a 62.5 micromolar solution of the positive control Cardamonin in H-Cort. medium, and add to row B of the 8-row dilution reservoir.Prepare a 06.25 percent solution of DMSO in H-Cort. meduim, and add to row C of the same reservoir. DMSO is used as the vehicle control.Use the automated liquid handler to remove 10 microliters of medium from wells B7 to B11 and C7 to C11 of each cell plate, and discard it into rows G and H of the reservoir. Be sure to use pre-configured tip boxes to address only those wells that will receive the positive and vehicle controls, and use a 50 microliter per second pipetting speed for all steps. Transfer 10 microliters of the positive and vehicle controls to those wells, and mix three times.Return these cell plates to the incubator. Repeat these steps for all additional cell plates. Perform all chemical exposure operations in a chemical fume hood, wearing double nitrile gloves, a laboratory coat, disposable polyethylene sleeve protectors, and safety glasses.Decontaminate pipette tips and reservoirs that have come into contact with the hydrofluoric acid, and any liquid hydrofluoric acid with 2.5 percent calcium gluconate prior to disposal in the hazardous waste stream. For each library plate under investigation, prepare a 0.0036 percent hydrofluoric acid medium solution by adding 288 microliters of one percent hydrofluoric acid to 80 milliliters of pre-warmed H-Cort. medium in a 150 milliliter bottle.Swirl the bottle, and incubate the dilute hydrofluoric acid in a 37 degree incubator in the chemical fume hood for ten minutes. Add the hydrofluoric acid medium solution to a reagent reservoir on a plate warmer. Perform the exposure with two technicians working in tandem.Have the technician on the right remove the cell culture medium from the cell plate using a twelve-channel pipetter, and then pass the plate to the technician on the left. Have the technician on the left add 100 microliters per well of the hydrofluoric acid medium solution. Repeat this process for all cell plates that are to be exposed to toxicant.Also, repeat this process for unexposed control plates, utilizing fresh medium instead of the hydrofluoric acid solution. Place the plates in a chemical fume hood incubator for 20 minutes, and then return them to the cell culture incubator. Repeat the positive control addition step as previously shown, once all plates have been exposed and returned to the cell culture incubator.Collect the cell culture supernatant and evaluate cell viability using an MTT assay 24 hours post-exposure. Prepare a solution of 0.5 milligrams per milliliter MTT substrate in PBS containing 10 grams per liter glucose, and warm to 37 degrees. Add the MTT substrate solution to a reservoir on an automated liquid handler.Use a 50 microliter per second pipetting speed for all steps. Pre-configure tip boxes to address only those wells to be assayed. Use the automated liquid handler to aspirate 95 microliters of medium from the inner 60 wells of the cell plate, and deposit 42.5 microliters into each of two 384-well storage plates.Immediately add 100 microliters per well of the MTT substrate solution to the cell plates, and incubate them at 37 degrees in a cell culture incubator for one hour. Seal the 384-well storage plates, and store them at minus 80 for subsequent analysis. Repeat this process for all top and bottom plate sets and the associated unexposed controls.Refill the MTT substrate reservoir as needed. Reuse tips between replicates if desired, but wash them when adding MTT substrate solution and when switching between plate sets. After the one hour incubation, use the automated liquid handler to remove the MTT substrate solution from the inner 60 wells of each cell culture plate, and replace with 100 microliters per well of DMSO.Use a 50 microliter per second pipetting speed for all steps, and pre-configure tip boxes to address only those wells to be assayed. Empty the waste reservoir and refill the DMSO reservoir as needed. Shake the plates on a plate shaker for three minutes.Measure absorbance at 570 and 690 nanometers, using a plate spectrophotometer. Subtract the background absorbance at 690 nanometers from the absorbance at 570 nanometers, and average the values for each control group on the unexposed plate. Calculate the percent viability by dividing the absorbance values for each target by the average absorbance value of the unexposed controls.Use the average unexposed negative pool to calculate the percent cell viability for all targets. Measure the concentration of IL-8 in cell culture supernatants using a no-wash bead-based assay, according to the manufacturer's instructions. Create an assay plate layout to accommodate samples and standards.Remove the 384-well storage plates from the minus 80 freezer, and thaw at room temperature. Briefly centrifuge the plates to collect the sample in the bottom of the wells. Add the anti-IL-8 acceptor beads and biotinylated antibody to a black 384-well storage plate.Add the bead-antibody mixture to the wells designated for use for samples and standard curve, according to the assay plate layout. Reconstitute the standard in medium containing 354 micromolar sodium chloride. Make twofold serial dilutions of the standard curve, and add in triplicate to a different black 384-well storage plate.Use an automated liquid handler to perform the assay. Use pre-configured tip boxes to address wells according to the assay plate layout. Use a 50 microliter per second pipetting speed for all steps.Transfer 8 microliters of the acceptor bead-antibody mixture to white 384-well shallow well assay plates. Add the bead-antibody mixture to wells designated for use for samples and standard curve according to the assay plate layout. Then, transfer the standard curve to the appropriate wells on the shallow well assay plates.Next, prepare a 3.54 molar sodium chloride solution in a shallow reservoir. Adjust the salt concentration of the samples to match that of the standard curve by transferring 4.5 microliters of the sodium chloride solution to the samples in the black 384-well storage plates. Mix the samples three times using a 30 microliter mix volume, and then transfer two microliters of the samples to the white shallow well assay plates according to the assay plate layout.Incubate for one hour in the dark at room temperature. Add secondary beads to a black 384-well storage plate. Add the beads to the wells designated for use for samples and standard curve according to the assay plate layout.Transfer 10 microliters of the secondary beads to wells of the white 384-well shallow well assay plate that will receive standard curve or samples according to the assay plate layout. Wash the tips between each plate. Incubate for another hour in the dark at room temperature.Seal the assay plates with clear plate seals, and scan using a plate reader compatible with the bead-based assay. Use a 0.2 millimeter distance between plate and detector, 180 millisecond excitation time for the scan, and 550 millisecond measurement time. Import the raw data from the IL-8 assays into a spreadsheet.Use automated curve fitting for the standard curve of the IL-8 assays, and then convert the raw data to picograms per milliliter for each sample. Calculate the average of the six replicates for each target. Then, calculate the SSMD values and fold change relative to the exposed negative controls.Display the data in a dual flashlight plot, which shows the fold change along the x axis and SSMD along the y axis, for each target. In conclusion, we have developed methods and culture conditions suitable for the high throughput screening study of ocular injury by toxicants. We have proven the utility of these methods by completing the primary screen of an siRNA library in a model of hydrofluoric acid injury.High throughput genomic data sets can greatly contribute to the understanding of the role that specific genes play in injury or disease, which can help more efficiently focus follow-on in vitro or in vivo studies.

Research

JoVE Journal

Biology

High-throughput Screening and Biosensing with Fluorescent C. elegans Strains

High-throughput Screening and Biosensing with Fluorescent C. elegans Strains

High-throughput screening (HTS) is a powerful approach for identifying chemical modulators of biological processes.  However, many compounds identified in ...

High-throughput Yeast Plasmid Overexpression Screen

The budding yeast, Saccharomyces cerevisiae, is a powerful model system for defining fundamental mechanisms of many important cellular processes, ...

High Throughput Screening of Fungal Endoglucanase Activity in Escherichia coli

High Throughput Screening of Fungal Endoglucanase Activity in Escherichia coli

Cellulase enzymes (endoglucanases, cellobiohydrolases, and &beta;-glucosidases) hydrolyze cellulose into component sugars, which in turn can be converted ...

High-throughput Screening for Small-molecule Modulators of Inward Rectifier Potassium Channels

Specific members of the inward rectifier potassium (Kir) channel family are postulated drug targets for a variety of disorders, including hypertension, ...

High-throughput Functional Screening using a Homemade Dual-glow Luciferase Assay

We present a rapid and inexpensive high-throughput screening protocol to identify transcriptional regulators of alpha-synuclein, a gene associated with ...

Recovery of Adult Zebrafish Hearts for High-throughput Applications

Use of the zebrafish model system for studying development, regeneration, and disease is expanding toward use of adult hearts for cell dissociation and ...

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 ...

Identification of Kinase-substrate Pairs Using High Throughput Screening


We have developed a screening platform to identify dedicated human protein kinases for phosphorylated substrates which can be used to elucidate novel ...

Multi-enzyme Screening Using a High-throughput Genetic Enzyme Screening System

The recent development of a high-throughput single-cell assay technique enables the screening of novel enzymes based on functional activities from a ...