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

Summary

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.

Abstract

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.

Introduction

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-epithelialized^1,2. Despite the best current therapeutic treatment options available, long-term prognosis is generally poor due to progressive corneal clouding and neovascularization^2,3.

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 CYP1A1^7,8.

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 throughput^11,12,13. 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 exposure^9,14. 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 outcome^2,18.

Protocol

1. Cell Culture Maintenance

1. Grow the cell line SV40-HCEC at 37 °C, 5% CO₂, and 90% humidity in DMEM F-12 with 15% fetal bovine serum (FBS), 1% L-glutamine, 10 µg/L epidermal growth factor (EGF), and 5 mg/L insulin.
2. Passage the cell line every 3 to 4 days (depending on seeding density) to ensure that confluency never exceeds 80% during culture maintenance.
3. Detach the cells from flasks using a detachment solution (14 mL solution for every 150 cm² flask) and incubation at 37 °C for no more than 8 min.
4. Neutralize the detachment solution with an equal volume of cell culture medium and pellet the cells in 50 mL conical tubes by centrifugation for 7 min at 160 x g.
5. Re-suspend the cell pellet in medium (20 mL for every 150 cm² flask).
6. Count the cells with an automated handheld cell counter and seed in new flasks at a density that will allow them to grow for 3 to 4 days without exceeding 80% confluency.

2. Plate Cells for Experimentation

1. Check flasks of SV40-HCECs by phase contrast light microscopy to ensure that confluency is less than 80% and to assess general cell health.
2. Detach, pellet, resuspend, and count SV40-HCECs from flasks as described in steps 1.3 to 1.6 of cell culture maintenance. Use SV40-HCEC medium containing 0.5 µg/mL hydrocortisone (HCORT medium) to resuspend cells.
3. In a disposable medium bottle, prepare a suspension of SV40-HCECs at 17,857 cells/mL in pre-warmed HCORT medium.
   1. Prepare a sufficient volume of cell suspension for the number of plates to be seeded.
   2. Seed a minimum of 14 x 96-well plates with cells for every siRNA library plate to be studied.
4. Swirl the bottle to evenly suspend the cells, pour a sufficient volume of the cell suspension into a reservoir on the orbital shaker nest of an automated liquid handler, and store the bottle containing the cell suspension on a 37 °C warming plate during the cell plating process.
5. Use the automated liquid handler to add cells to plates at a density of 1000 cells per well (5000 cells/cm²) in 70 µL of medium per well of a 96-well plate. Use a 50 µL/s pipetting speed for all steps.
   1. Run the orbital shaker at 100 rpm constantly during the seeding process.
   2. Mix the cell suspension three times (140 µL mix volume) using the automated liquid handler.
   3. Aspirate 140 µL of the cell suspension and dispense 70 µL into each well of two cell culture plates.
   4. Repeat steps 2.5.3 and 2.5.4 until all plates have been seeded with cells.
   5. Refill the cell suspension reservoir as needed during the seeding process.
6. After the plates have been seeded, remove them from the automated liquid handler and incubate them for 30 min at room temperature before transferring them to a cell culture incubator.
7. Evaluate the cell density of every well for each plate the following morning using an automated imaging system which is housed in a cell culture incubator and exclude any plates from the study which have interior wells that are not between 15% and 22% confluent.

3. Transfect Cells with siRNA

1. Acquire siRNA library plates from the vendor pre-configured to contain 80 siRNA targets per plate (see Figure 4), with columns 1 and 12 left empty.
2. Reconstitute the siRNA library plate with siRNA buffer to a final concentration of 2 pmol/µL for each well of siRNA according to the manufacturer's instructions¹⁹.
3. Transfect the cells 24 h after seeding with 4 pmol/well of siRNA and 0.3 µL/well of transfection reagent. Perform all steps according to the transfection reagent manufacturer's protocol (see Figure 4 for plate layout)²⁰.
   1. Perform all the transfections using an automated liquid handler, and use a 25 µL/s pipetting speed for all steps. Use pre-configured tip boxes to address only the wells that will be transfected.
   2. Use the top half of the library plate to transfect a set of six replicate plates referred to as the "Top Plate Set" (six replicates per siRNA, n=6).
   3. Use the bottom half of the library plate to transfect a different set of six replicate plates referred to as the "Bottom Plate Set" (six replicates per siRNA, n=6).
   4. Use the term "Full Plate Set" to describe the 12 cell plates that have been transfected with library siRNA.
   5. Transfect wells B2-B6 of all plates in the Full Plate Set with negative pool siRNA after all top and bottom plate sets have been transfected with library siRNA.
   6. Also transfect wells B2-B6 of another two plates, that do not receive library siRNA and will serve as unexposed controls (one for the top plate set and one for the bottom plate set), with negative pool siRNA.
4. Incubate all cell plates in a cell culture incubator for 4 hours after all transfection mixes have been added and mix by gentle tapping every hour.
5. Use an automated liquid handler to wash all wells of the plates twice with pre-warmed HCORT medium diluted 1:5 in PBS. Use a 50 µL/s pipetting speed for all steps.
   1. Aspirate 100 µL from each well and discard that into a waste reservoir.
   2. Add to each well 100 µL/well pre-warmed HCORT medium diluted 1:5 in PBS which is contained in a different reservoir at a sufficient volume of for the number of plates.
   3. Repeat steps 3.5.1 and 3.5.2 for each plate.
      1. Empty the waste reservoir as needed.
      2. Refill the reservoir with HCORT medium diluted 1:5 in PBS as needed.
   4. Aspirate 100 µL from each well and discard that into the waste reservoir.
6. Refeed all wells of the plates with 100 µL/well pre-warmed HCORT medium, which is contained in a different reservoir at a sufficient volume of medium for the number of plates.
   1. Refill the reservoir with medium as needed.
7. Use wells C2-C6 of all plates as non-transfected controls.
8. Keep wells B7-B11 and C7-C11 of all plates non-transfected to be used as drug and vehicle controls for toxicant exposure.

4. Refeed the Cells the Following Day

1. Use an automated liquid handler to refeed all wells of the plates. Use a 50 µL/s pipetting speed for all steps.
   1. Aspirate 100 µL from each well and discard that into a waste reservoir.
   2. Refeed each well with 100 µL/well pre-warmed HCORT medium that is contained in a different reservoir and has a sufficient volume of medium for the number of plates to be refed.
   3. Repeat steps 4.1.1 and 4.1.2 as needed to refeed all cell plates.
      1. Empty the waste reservoir as needed.
      2. Refill the reservoir with medium as needed.

5. Positive Control Addition

1. Two days after transfection and two hours prior to exposure, prepare a 62.5 µM solution of the positive control cardamonin in HCORT medium to be used for HFA exposures and add to row B of an 8-row dilution reservoir.
   1. For CP exposure, prepare and use a 25 µM solution of SKF 86002.
   2. Prepare a volume of positive control sufficient for the number of plates to be tested.
2. Prepare a 0.625% solution of DMSO in HCORT medium (vehicle control) and add to row C of the same reservoir.
   1. For CP exposure, prepare and use a 0.5% solution of DMSO.
   2. Prepare a volume of vehicle control sufficient for the number of plates to be tested.
3. Use the automated liquid handler to add positive and vehicle controls to wells B7-B11 and C7-C11 of each cell plate and use a 50 µL/s pipetting speed for all steps. Use pre-configured tip boxes to address only the wells that will receive the positive and vehicle controls.
   1. Remove 10 µL of medium from wells B7-B11 and C7-C11 of each cell plate and discard it into rows G and H of the 8-row dilution reservoir.
   2. Transfer 10 µL of the positive and vehicle controls to those wells and mix 3 times.
4. Return these cell plates to the incubator.
5. Repeat steps 5.3 to 5.4 until all cell plates have received positive and vehicle controls.

6. HF Exposure of Cultured Cells

CAUTION: HFA is corrosive and acutely toxic.

1. Perform all chemical exposure operations in a chemical fume hood wearing double nitrile gloves, a laboratory coat, disposable polyethylene sleeve protectors and safety glasses.
   1. Acquire HFA as a 48% solution.
   2. Dilute HFA to 1% with ultrapure water and store in 5 mL aliquots in 10 mL thick wall polyethylene vials to improve safety.
   3. Decontaminate pipette tips and reservoirs that have come into contact with HFA and any leftover liquid HFA with 2.5% calcium gluconate prior to disposal in the hazardous waste stream.
2. For each siRNA library plate under investigation, prepare a 0.0036% HFA medium solution by adding 288 µL of 1% HFA to 80 mL of pre-warmed HCORT medium in a 150 mL bottle.
   1. Swirl the bottle and incubate the dilute HFA in a 37 °C incubator in the chemical fume hood for 10 min.
3. Mix the HFA medium solution again by swirling the bottle and add the HFA medium solution to a reagent reservoir on a plate warmer.
4. Perform the exposure with two technicians working in tandem.
   1. Have the technician on the right aspirate 100 µL from each of the interior wells of a cell plate using a 12 channel pipettor and then pass the plate to the technician on the left.
   2. Have the technician on the left add 100 µL per well of the HFA medium solution.
   3. Repeat steps 6.4.1 and 6.4.2 for all cell plates that are to be exposed to toxicant.
   4. Also repeat steps 6.4.1 and 6.4.2 for unexposed control plates, utilizing fresh HCORT medium instead of the HFA medium solution.
5. Place the plates in the chemical fume hood incubator for 20 min and then return them to the cell culture incubator.
6. Repeat the positive control addition step (steps 5.1 to 5.5) as previously shown once all plates have been exposed and returned to the cell culture incubator.

7. CP Exposure of Cultured Cells

CAUTION: CP is acutely toxic and an irritant.

1. Perform all chemical exposure operations in a chemical fume hood wearing double nitrile gloves, a laboratory coat, disposable polyethylene sleeve protectors and safety glasses.
   1. Acquire CP.
   2. Dilute CP to 5% in DMSO and store in 10 mL aliquots in 10 mL scintillation vials to improve safety.
   3. Decontaminate pipette tips and reservoirs that have come into contact with CP and any leftover liquid CP with 2.5% sodium bisulfite prior to disposal in the hazardous waste stream.
2. Prepare a sufficient volume of pre-warmed HCORT medium containing 1x Pen/Strep and pre-warmed PBS for the number of plates to be exposed.
3. For each Top Plate Set or Bottom Plate set, add 8.04 µL of 5% CP in DMSO to a 50 mL aliquot of pre-warmed HCORT medium and mix well for a final CP concentration of 0.0008%. Cap the tube tightly and incubate the solution in a 37 °C incubator in the chemical fume hood for 1 h.
   1. Time the addition of CP to the 50 mL aliquots of medium so that each exposed Top Plate Set and Bottom Plate Set receives toxicant exactly 1 h after CP was added to the 50 mL aliquot of medium.
4. Remove a Top Plate Set and 1 unexposed control plate from the cell culture incubator and place them in the chemical fume hood near the end of the incubation period.
5. Mix the CP solution by inverting the tube and then decant it to a reagent reservoir at the end of the 1 h incubation period.
6. Perform the exposure with two technicians working in tandem.
   1. Have the technician on the right aspirate 100 µL from each of the interior wells of a cell plate using a 12 channel pipettor and then pass the plate to the technician on the left.
   2. Have the technician on the left add 100 µL per well of the CP medium solution.
   3. Repeat steps 7.6.1 and 7.6.2 for all cell plates of the Top Plate Set to be exposed to toxicant.
   4. Also repeat steps 7.6.1 and 7.6.2 for unexposed control plates, utilizing fresh HCORT medium instead of the CP medium solution.
7. Place the plates in a 37 °C incubator in the chemical fume hood for 10 min.
8. Add a sufficient volume of PBS and HCORT medium containing 1x Pen/Strep to reagent reservoirs near the end of the 10 min incubation period.
9. Remove the CP solution from cell plates using a 12 channel pipettor and replace it with 100 µL per well of PBS at the end of the 10 min incubation period.
10. Immediately remove the PBS from cell plates and replace it with 100 µL per well of HCORT medium containing 1x Pen/Strep.
11. Also repeat steps 7.9 and 7.10 for the unexposed control plates.
12. Return the cell plates to a standard cell culture incubator.
13. Repeat steps 7.3 to 7.12 for the Bottom Plate Set.
14. Repeat the Positive control addition step (steps 5.1 to 5.5) as previously described once all plates have been exposed and returned to the cell culture incubator.

8. Sample Collection and Cell Viability Assay

1. Twenty four hours after exposure, prepare a solution of 0.5 mg/mL MTT substrate in PBS containing 10 g/L glucose and warm to 37 °C²¹. Prepare 10 mL of substrate for each plate to be assayed.
2. For the number of plates to be assayed, add a sufficient volume of MTT substrate solution to a reservoir on an automated liquid handler.
3. Use the automated liquid handler to collect sample and add MTT substrate using a 50 µL/s pipetting speed for all steps. Preconfigure tip boxes to address only those wells to be assayed.
   1. Aspirate 95 µL of medium from the inner 60 wells of the cell plate, and deposit 42.5 µL into each of two 384-well storage plates.
   2. Immediately add 100 µL per well of the MTT substrate solution to the cell plates, and incubate them at 37 °C in a cell culture incubator for 1.5 h.
   3. Refill the reservoir with MTT substrate solution as needed.
4. Incubate the cell plates at 37 °C in a cell culture incubator for 1 h.
5. Seal the 384-well storage plates and store them at -80 °C for subsequent analysis.
6. Repeat steps 8.3 to 8.5 for all top and bottom plate sets and the associated unexposed controls.
   1. Reuse tips between replicates if desired, but wash them when adding MTT substrate solution and when switching between plate sets.
7. After the 1 h incubation, add a sufficient volume of DMSO to a reservoir on an automated liquid handler.
8. Use the automated liquid handler to add DMSO and use a 50 µL/s pipetting speed for all steps.
   1. Aspirate 100 µL from each of the inner wells of the cell plates and discard the MTT substrate solution into a waste reservoir.
      1. Empty the waste reservoir as needed.
   2. Overlay each well with 100 µL DMSO.
      1. Refill the DMSO reservoir as needed.
9. Shake the plates on a plate shaker for 3 min.
10. Measure absorbance at 570 and 690 nm using a plate spectrophotometer.
11. Subtract the background and calculate the % cell viability by dividing the exposed absorbance values by the unexposed controls. Use the average unexposed negative pool siRNA control to calculate % cell viability for all targets.

9. Measure IL-8 Concentration in Cell Culture Supernatants

1. Measure the concentration of IL-8 in the cell culture supernatants using a no wash bead-based assay according to the manufacturer's instructions²². Create an assay plate layout to accommodate samples and standard curve.
2. Remove the 384-well storage plates from the -80 °C freezer, thaw at room temperature, and briefly centrifuge the plates to collect the sample in the bottom of the wells.
3. For the number of assay plates to be run, make a sufficient volume of anti-IL-8 acceptor beads and biotinylated anti-IL-8 antibody in assay buffer included in the kit. Use the ratio of 50 µL of anti-IL8 acceptor beads and 50 µL of biotinylated anti-IL8 antibody to 9.9 mL of assay buffer.
   1. Using a multichannel pipettor, add bead/antibody mixture to a black 384-well storage plate.
      1. Add acceptor bead/antibody to the wells designated for use for samples and standard curve according to the assay plate layout.
      2. Add a sufficient volume per well for the number of assay plates to be run.
4. Reconstitute IL-8 standard to 1000 pg/mL using cell culture medium containing 354 µM NaCl. Make a sufficient volume for the number of assay plates to be run.
5. Make eight twofold serial dilutions of the standard curve.
6. Add the standard curve in triplicate to the appropriate wells of a black 384-well storage plate according to the assay plate layout. Add a sufficient volume per well for the number of assay plates to be run.
   1. Similarly, add cell culture medium containing 354 µM NaCl with no IL-8 for background measurement.
7. Use an automated liquid handler to perform the assay. Use pre-configured tip boxes to address only the wells designated by the assay plate layout. Use a 50 µL/s pipetting speed for all steps.
   1. Transfer 8 µL of the acceptor bead-antibody mixture to the wells of the white 384-well shallow well assay plates.
      1. Add only to the wells designated for samples and standard curve according to the assay plate layout.
   2. Transfer 2 µL of the standard curve to the appropriate wells of the shallow well assay plates according to the assay plate layout.
   3. Prepare a 3.54 M NaCl solution and add a sufficient volume for the number of plates to be assayed to a shallow reservoir on the automated liquid handler.
   4. Adjust the salt concentration of the samples to match that of the standard curve by transferring 4.5 µL of the NaCl solution to the samples in the black 384-well storage plates.
   5. Mix the samples three times using a mixing volume of 30 µL, and transfer 2 µL of the samples to the white shallow well assay plates according to the assay plate layout.
      1. Change tips in between sample plates.
   6. Incubate for 1 h in the dark at room temperature.
   7. For the number of assay plates to be run, make a sufficient volume of acceptor beads in assay buffer. Use the ratio of 200 µL of secondary acceptor beads to 12.3 mL of assay buffer.
   8. Using a multichannel pipettor, add secondary beads to a black 384-well storage plate.
      1. Add secondary beads to the wells designated for use for samples and standard curve according to the assay plate layout.
      2. Add a sufficient volume per well for the number of assay plates to be run.
   9. Using the automated liquid handler, transfer 10 µL of the secondary beads to the wells of the white 384-well shallow well assay plates.
      1. Add only to the wells that received samples and standard curve according to the assay plate layout and wash the tips between each plate.
   10. Incubate for another hour in the dark at room temperature.
8. Seal the assay plates with clear plate seals and scan using a plate reader compatible with the assay. Use 0.2 mm distance between plate and detector, 180 ms excitation time, and 550 ms measurement time for the scan.
9. Import the raw data from the IL-8 assays into a spreadsheet.
10. Use automated curve fitting for the standard curve of the IL-8 assays, and then convert the raw data to pg/mL for each sample.

Results

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...

Discussion

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...

Materials

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