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.
