Keep your HCA under control

Having sufficient replicates is not enough to ensure reliable results. An important but commonly overlooked aspect of image-based assay design for high content analysis (HCA) is the use of appropriate controls.

It is critical to use the correct controls for every run, plate and experiment with image-based data. For image-based assays, there are two types of control — treatment controls and labelling controls.

Treatment controls

The use of treatment controls (also known as experimental controls) allows you to correctly assess the effects of your compound of interest. For assessing compounds, you should always include three types of controls:

• Positive control: fluorescently labelled cells in the presence of a treatment with a known effect. This allows for assessment of changes with the biology, including problems with the cells you are using. This also allows you to evaluate your staining reagents, and to adjust your exposure settings appropriately. The use of positive controls is often overlooked, but it is extremely important for ensuring correct biological responses. When cells do not respond to the positive, it can indicate problems with the biology, plate or sample preparation.
• Negative control: fluorescently labelled cells without any treatment. The role of the negative control is to inform the user about the native state of the cells without treatment, and to demonstrate that effects seen in experimental wells are due to treatment with your compound of interest. It can also be compared to the vehicle control.
• Vehicle control: fluorescently labelled cells with only the substance in which your compounds are dissolved. This allows you to assess whether the changes you are seeing are due to the vehicle (for example, DMSO or ethanol) or the compound of interest. The vehicle control demonstrates that the vehicle (what your compound is dissolved in) does not elicit the response.

Figure 1: Compound controls in high content assays. Positive controls are used to elicit a known response, and to help determine that the cells/biology are responding appropriately. The negative control is used to understand the background levels of staining, and the native state without treatment. The vehicle-only control confirms that the responses observed following treatment with compounds are not due to the vehicle (the substance that the compound is being dissolved in). Secondary-only controls are used to ensure that there is no non-specific binding of the secondary antibody to the sample. This non-specific binding can be caused by insufficient blocking during staining. The autofluorescence control is used to ensure that the sample itself (cells, media, plate) is not emitting large amounts of autofluorescence that could interfere with the assay. For a larger image, click here.

Labelling controls

In addition to experimental controls, you should also include labelling controls. Labelling controls include the following:

• Secondary-only control: cells with secondary antibodies only. The secondary-only control is used to ensure that any secondary antibodies are not exhibiting non-specific binding in the assay. This non-specific binding can often be attributed to insufficient blocking during the antibody staining protocol.
• Autofluorescence control: unlabelled cells. The autofluorescence control is used to detect background fluorescence of the sample. This could be autofluorescence of the cells or the media, and helps you determine what is background and what is true signal.
   

By taking the time up front to set up the correct controls, you can get better answers from your HCA experiments, faster and with greater confidence.

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Top image credit: ©iStockphoto.com/Sebastian Kaulitzki