Protocol

Preparation of Cells in 96-Well Plates for siRNA Transfection and High-Content Analysis

This protocol was adapted from "High-Content and High-Throughput Screening," Chapter 13, in Cell Imaging (ed. Stephens). Scion Publishing Ltd., Oxfordshire, UK, 2006.

INTRODUCTION

RNA interference using siRNA libraries is a powerful technology for elucidating gene function by downregulating gene expression at the post-transcriptional level. Phenotypic changes associated with siRNA knockdown can be monitored using cell lines expressing fluorescent reporter proteins. Test siRNAs are transiently transfected into the reporter cell line and behavior of the fluorescent reporter probe is monitored using a high-content imaging system. A range of cell features and additional fluorescent probes can be monitored to assess the effects of knockdown. This article details how to perform siRNA transfection in multiwell plates for subsequent high-content analysis.

RELATED INFORMATION

Protocols for Optimization of siRNA Knockdown in EGFP-Expressing Cell Lines Using High-Content Analysis and Analysis of siRNA Knockdown of Cell-Cycle Control Genes in G1/S and G2/M Cell-Cycle Phase Marker Cell Lines Using Multiplexed High-Content Analysis are also available.

MATERIALS

Reagents

Cell line of interest and appropriate culture medium

See Discussion for considerations regarding choice of cell line.

DharmaFECT cell-culture reagent (Dharmacon)

DharmaFECT transfection reagent (Dharmacon)

Choose a transfection reagent appropriate to the cell line based on manufacturer’s data or determine the optimal reagent and conditions as described in Optimization of siRNA Knockdown in EGFP-Expressing Cell Lines Using High-Content Analysis.

Fetal bovine serum (FBS) (Sigma)

Penicillin (Sigma)

Phosphate-buffered saline (PBS) (1X; sterile, calcium- and magnesium-free)

siARRAY RTF siRNA library (96-well plate; Dharmacon)

Streptomycin (Sigma)

Trypsin-EDTA (Sigma)

Equipment

Flasks for cell culture

Hemocytometer or disposable cell-counting chamber (Immune Systems)

Incubator, tissue culture (37°C, 5% CO₂, humidified)

Microscope, inverted

Pipette, multichannel

Plates, 96-well imaging-grade (e.g., microclear black [Greiner] or ViewPlate [Packard])

METHOD

1. Grow cells in flasks in an incubator in medium supplemented with 10% FBS, 100 U/mL penicillin, and 100 µg/mL streptomycin. Do not allow cells to exceed 90% confluency.

2. Equilibrate the siARRAY plate to room temperature.

3. For each well to be transfected, combine 0.125 µL of DharmaFECT transfection reagent with 24.875 µL of DharmaFECT cell-culture reagent and add 25 µL to each well of the siARRAY plate. Incubate for 20-90 min at room temperature to rehydrate the siRNAs.

4. Remove the culture medium from the cells, wash cells once with PBS, and add a small volume

(e.g., 5 mL) of trypsin-EDTA. Incubate at 37°C to detach the cells.

5. Dilute the cell suspension with an equal volume of medium with 10% FBS but without antibiotics.

6. Count the cell suspension and dilute to 5000-10,000 cells/100 µL in medium with 10% FBS but without antibiotics.
The optimum cell number will depend on the cell line and the intended duration of the experiment. Seeding at 5000-10,000 cells per well should give good results for common laboratory cell lines in knockdown experiments lasting 24-48 h.

7. Aliquot 100 µL of cell suspension to each well containing rehydrated siRNAs.

8. Using a multichannel pipette, immediately transfer the cell and siRNA mixtures from the siARRAY plate into equivalent wells in the imaging plate.
This step may be omitted if the siARRAY RTF products are supplied in plates compatible with imaging and high-content analysis.

9. Incubate the plate for the desired period before processing for imaging. For details about imaging and analysis, see Optimization of siRNA Knockdown in EGFP-Expressing Cell Lines Using High-Content Analysis.
See Troubleshooting.

TROUBLESHOOTING

Problem: Background is high and/or the analysis is unsuccessful.

[Step 9]

Solution: Take care to minimize cell disruption and avoid creating vortices when adding reagents to wells. Cells that are dead, dying, dividing, or blocked in mitosis may be weakly adherent and therefore particularly susceptible to disturbance. Selective loss of these cell subpopulations will skew the analysis results. To minimize background fluorescence, use the plates, serum, and culture medium from the recommended suppliers.

Problem: Image quality needs improvement.

[Step 9]

Solution: Inspect images to ensure that field illumination is even and that features to be quantified are bright and in focus. If not, adjust the instrument and acquisition set-up and reimage the samples. If reacquisition is not possible, try applying noise reduction or shading correction techniques before analysis. Overexposure of images can be as detrimental as underexposure. Check pixel gray-level values of representative cell features to ensure that they fall within the dynamic range of the camera. If not, reimage the sample using a shorter exposure time, decrease the laser power, or add a neutral-density filter.

DISCUSSION

When choosing a cell system for use with this protocol, some consideration should be given to the characteristic cell morphology and growth habits. Most detection platforms are designed to image and analyze cells growing in monolayers. Cells that tend to cluster together or grow on top of each other may be difficult to resolve and analyze. Likewise, cells that adhere loosely and take on spherical morphologies are usually more difficult to image and analyze than those that adhere well to the plate and spread out flat across its surface. When cells are relatively thick or rounded up due to poor adherence, various structures can lie in different planes of focus, which can lead to imaging problems. The method by which an imaging system finds optimal focus can affect how well the system performs on samples of varying thickness. The majority of high-content systems now employ laser-based hardware autofocus mechanisms that locate the well bottom and then focus a specified offset distance above this location.

How the cells are handled during maintenance and sample preparation will have a direct impact on assay quality. Harsh conditions can apply selection pressure to cell cultures, resulting in population heterogeneity and changes in phenotype, including loss of reporter expression. In our experience, it is advisable to passage cells while they are in exponential-phase growth, avoid seeding the cells at densities that are too sparse or too confluent, and discard them after ~25 passages. When preparing samples for screening, seeding density and evenness of distribution can be critical for minimizing variability. The use of automated cell-culture and sample preparation systems can reduce the time, cost, and labor involved in cell handling, and can help improve the consistency and accuracy of the assay results.

Cells may be imaged live or preserved with fixative prior to imaging. For higher-throughput screens, fixed-cell formats have a number of advantages over live-cell protocols. Fixed assay plates can be transported easily to the screening facility. Prior to screening, samples can be accumulated and stored for several weeks or months, making it easier to schedule screening runs. There is no need for environmental control during the screen, and temporal variability is reduced, leading to better assay signal-to-noise ratios. After screening, fixed samples can be returned to storage and then retrieved at a later date for reimaging if necessary. Some applications may not be amenable to fixation, or may perform better in a live-cell format. For example, reporter fluorescence or localization may not be preserved upon fixation, the cellular response may occur too rapidly to enable fixation, or the fixation process may introduce artifacts. Imaging the cells while they are alive can overcome these problems, as well as simplify the logistics of kinetic assays. For live-cell screens, environmental control can be critical for maintaining cell health and minimizing temporal variability throughout the duration of the screen. Most mammalian cell cultures require a humidified environment with a constant temperature of 37°C and 5% CO₂.

The use of validated commercial assays and reagents for cell-based screening can reduce the time and expense of assay development. Cellomics has developed a range of bioapplication kits that contain validated combinations of antibodies, fluorescent dyes, and other reagents. GE Healthcare offers a panel of cell lines stably expressing green fluorescent protein (GFP) reporter proteins that are involved in key cell-signaling pathways. The cell lines are well characterized and accompanied by validated assay protocols. In addition, the expression vectors used to establish the cell lines are provided so that customers can use them to genetically engineer cell lines of their choice.