Cell-based assays are widely used within drug development programs to assess the effects of test compounds on cellular targets, evaluate cytotoxicity, and monitor proliferation. While these assays do provide a biologically relevant system for predicting the human response to a potential drug candidate, several factors require careful consideration to ensure optimal assay performance and sensible interpretation of data. Vanessa Fonte, product manager at Enzo Life Sciences, shared with us her ten tips for cell-based assay success.
Cell-based assays regularly utilize continuous cell lines. These are often favored because they are easy to culture, yielding requisite cell numbers in a short timeframe. That being said, it is essential to confirm that the target is expressed at measurable levels during assay optimization. Although primary cells can be more challenging to culture and transfect, they may be preferred, particularly in cases where available cell lines have been found to be misidentified. When performing cytotoxicity assays, or viability assays that measure metabolite production, it should be considered that metabolic and apoptotic rates often differ between cell lines and primary cells.
Fresh media should always be used to ensure healthy cell growth. It is wise to check the growth requirements of your cells with the supplier, since altering media conditions may lead to changes in cellular behaviors. Any necessary supplements should also be freshly prepared, and if appropriate, should be stored as frozen aliquots to prolong their effective utility. All culture media and supplements should be purchased from a consistent source and a record detailing lot numbers should be maintained.
Regular servicing and maintenance will ensure optimal incubator performance, while daily monitoring of temperature and CO2 levels will allow any malfunctions to be identified and dealt with promptly. Keep the time for which the incubator door is left open to a minimum, don’t overfill the shelves, and avoid stacking plates to prevent edge effects.
It is good practice to disinfect your biosafety cabinet and all necessary materials before starting your experiment. Only place items that you need within the cabinet and avoid the air grilles to minimize obstructions that may block air flow. Wear appropriate personal protective equipment (PPE) and use only sterile media and consumables.
Cells are extremely delicate, so it is important that any pipetting is performed gently. Adherent lines should not be over-trypsinized, while extended passaging of any cell line should be avoided since genetic and phenotypic drift can occur over time, resulting in altered cellular characteristics.
Inaccurate pipetting is a major source of experimental error. You should confirm that your pipettes are calibrated before beginning work. If pipetting cells from a trough, mix them gently but thoroughly to prevent clumps and ensure an even distribution. Any reagents should also be carefully mixed.
Controls are essential to accurate data interpretation, which is why positive and negative controls should always be included. The nature of these will vary depending on the experiment, but they should afford a sensible assay window. It is also important to incorporate a standard curve into many cell-based assays.
Assay data will only be of relevance if the cells used are healthy. Don't passage cells continuously for extended periods of time, and never allow them to become over-confluent in flasks. The color of the growth media can often be used as an indicator that cells require splitting. Perform a viability count alongside the overall cell count before seeding cells for use.
Different cell-seeding densities should be compared during assay optimization to maximize the assay window. The cell number should be high enough to allow the production of a measurable signal, but over-crowding should always be avoided.
The choice of reader will vary depending on the nature of the assay. If your readout is fluorescent, check the compatibility of any filters to avoid spectral overlap, and select appropriate microplates to prevent fluorescent cross-talk. Microplates with clear bottoms and black sides are often chosen for fluorescent plate-based cellular assays. Colorimetric readouts can be prone to variability and high background due to user bias or reagent precipitation. That being said, advances in reagent development mean colorimetric readouts are still commonly in use, particularly for cell-based assays that measure proliferation or viability.
Cell-based assays are essential to research programs worldwide and continue to be pivotal to the process of drug development. By selecting appropriate cell lines, using suitable media and reagents, and by giving due consideration to assay setup and experimental technique, they will continue to provide reliable and high-quality data for years to come.