Often times we get questions about cell based assays (bioassays) that seemed like they should have performed better than they did. Since we perform bioassays all the time, we have developed an awareness of the things that can make an assay perform even better. Here are five.

Cell Morphology
Knowing your cells and how they look at each stage is essential to being able to recognize the lag, log and plateau stages. Any change in cell morphology can be a change in their metabolism. One example of transient morphological changes was with a Rauscher erythroleukemia line. We found that if the cultures were “jostled” just by carefully moving the flasks from the incubator to the microscope, transient blebs/protrusions would form. However, if we allowed the flask to remain stationary on the microscope stage for ~15 minutes the blebs would disappear, and the cells would become spherical. Another example we found that some cells change their appearance and develop granules when they approach plateau concentration. Both these examples could translate into a changing metabolism that could affect your assay. The best practice is to add your drug to the cultures when they appear “normal”, without blebs, in log growth. Normal is different for many cell lines, therefore knowing your cells through microscopic observation allows you to make informed decisions that can be replicated from assay to assay and provide a robustness otherwise not achieved.

Culture Time
Some assays have cultured cells that require 7 or 8 days or longer. During that extended time evaporation may become a factor, even if the outer wells contain media/water or PBS. There will be some evaporation. This evaporation will change the concentration of salt as well as other media components. In some of these long-term cultures we place the plates into a hydration chamber that allows for CO2 permeation, and traps in the water vapor, further minimizing evaporation. We have found far better outcomes using this approach when compared to using a shelf above the incubator’s water reservoir. Additionally, in 96 well plates we use the maximum volume per well, 200uL, which decreases the effect of evaporation compared to a 100uL volume, even for overnight incubations. If needed, we re-feed the cultures starting by withdrawing media, up to 150uL, then adding a fresh aliquot, even though this introduces a variable that could be reflected in the final assay data. Some media components are labile over time at 37oC, and some cells are sensitive to this deterioration of the media. Refeeding has a rejuvenation effect which is a good trade-off with the increased variability in the data.

Cell Plating
In proliferation or in “cell death” assays, it is imperative that adherent cells are evenly spaced throughout the bottom surface. Just moving the 96 well assay plate from the hood to the incubator sets up a circular vortex of the media whereby the cells move to the outer edge of the well. This results in cells piling on top of each other along the perimeter of the well with a “bald” center. If this occurs the cell’s access to nutrients and your drug is variable and is reflected in the results. Additionally, specific to proliferation assays, if the cells are piled on top of each other, there will be differences in the “space to grow to gain a foothold onto the plastic” and will contribute to greater variation in the results. Typically, we allow the cells to slightly adhere before we move the plate from the hood to the incubator, keeping an “eagle eye” on the pH of media. You don’t want the media to become basic. At each step of the assay, microscopic observation of a reasonable number of wells is essential to insure there are no irregularities in the cultures that would yield to poor quantitative data.

Substrate Development Time
Any assay for which color/fluorescence/luminescence development is a part, our timing for substrate development is typically 20-30 minutes with an incubation temperature of 37oC, since most enzymes have maximal activity at 37oC. When a shorter incubation time is necessary (~10 minutes), we may use room temperature to avoid taking a plate in and out of a 37oC incubator which can create uneven temperature gradients within the plate, resulting in variation of the precision of the assay (e.g. plate-to-plate, day-to-day). If room temperature is used, the enzyme concentration (e.g. horse radish peroxidase) is changed since it is at sub-maximal activity.

Drug Concentration Curve
When we are developing a 4-parameter drug concentration curve, we anchor each plateau far from the inflection point, which prevents the curve from moving from day-to-day. We use concentrations that describe the two inflection points between which the linear portion results in a precise EC50 determination.

These are just a few tidbits that our experience in assay development has led us to control. Hopefully this helps you in your assay development.