Balancing Assay Speed, Reading Sensitivity, and Culture Conditions
Interfering events wreak havoc with live assays. Cells’ natural fluorescence overlapping with detection signals is one example. Moreover, auto fluorescence varies unpredictably with cell health, which is a function of temperature, humidity, and gas composition.
As a consequence of the interplay among those variables, sensitivity and consistency may vary significantly across a microplate. Dying cells in one location could reduce or even enhance apparent sensitivity. If the assay is based on a metabolic process, having fewer cells will automatically reduce available signal. “Detecting above background noise is always more difficult when cells are not healthy and happy,” says Carl Peters, PhD, applications scientist at BMG LABTECH (Offenburg, Germany).
Controlling for cell health
Cell health is not much of an issue during minutes-long experiments. Problems arise with the need to maintain cell health over many hours or days. Reliably discriminating signal from noise in those situations requires environmental control, often with an independent measure of cell health during the elapsed time of the assay.
“But even when you control for cell health, other factors can easily affect sensitivity,” Peters tells Lab Manager.
Maintaining healthy cells involves adding components to readers or robotics that mimic conditions inside an incubator. Most readers control temperature, but maintaining carbon dioxide, oxygen, and humidity levels is difficult without lidding. However, covering and uncovering exposes cells to ambient conditions and adds a step, whether operations are fully automated or manual.
“We have not been able to come up with a way to maintain a humidified environment within a microplate reader,” Peters admits. “Plate sealers and lidding will reduce evaporation but not eliminate it.”
An operational approach for minimizing environmental exposure is to adopt assays with reduced reading time or more-sensitive readers. Switching assay readouts may provide faster reading and less interference from native fluorescence, thereby improving sensitivity. More advanced assays like FRET (fluorescence resonance energy transfer) or offshoots may provide greater speed and sensitivity while eliminating troublesome background signals.
“If you’re in this for the long haul, setting up the appropriate assay for your outcome is time well spent,” Peters says.
Maximizing incubator time
There are two major types of cell-based assays. In endpoint assays, experimenters lyse cells to release their contents and quantify what is inside—say, a protein or a strand of DNA. Live cell assays monitor cell activity in real time. Both assay types require healthy, functioning cells, but for different reasons and durations.
Live cell assays require juggling numerous factors to ensure cells are working properly, according to Peter Banks, PhD, scientific director at BioTek Instruments (Winooski, VT). One goal of plate handlers and experimental design is to minimize the time cells spend outside the incubator. While the plate is on the reader deck, cell health must be maintained either through built-in capabilities or with the assistance of add-on components. Vendors have succeeded for the most part in achieving this. “Today it is possible to perform longterm kinetic studies with the plate on the reader deck,” Banks says.
One strategy for long-term kinetic evaluations is discontinuous kinetic read. Plates are read at closely spaced time points at the beginning, but at the end are returned to incubators for readings that are more spread out. This, Banks says, allows extending assay time without needing to take data points every few minutes. “Top readers will allow rapid measurements at the beginning of a study [when] kinetics are fast, then will stagger readings—perhaps every hour or even every day.” Plates leave the incubator only for washes, media replacement, and readings.
Banks claims to have run weeks-long assays in this manner. “With the assistance of fast, sensitive readers, cells spend nearly all their time inside the incubator,” he says. “Medium exchanges or feeds occurring every few days take just a few minutes.” When plates are inside incubators, gas-permeable lids allow the exchange of oxygen and carbon dioxide while minimizing evaporation.
Cell-based assays share many of the workflow components of simpler biochemical assays, but differences in plate storage, agitation, and reading support the special needs of living organisms. Workflows and automation should be engineered to minimize the time cells spend on the reader deck, as well as the duration of fluid exchanges and reagent addition.
Formats for microplate wells are relatively unimportant for non-cell assays, but medium/buffer exchanges for longduration cell-based assays require a tilting rack to facilitate complete removal of media. “Remaining buffer levels would be too high with center-only orientation, which would dilute relevant reagents as well as the detection range,” says Susanne Braum, PhD, senior product manager for liquid handling and cell biology at Tecan (Männedorf, Switzerland).
Cell-based assays requiring long reads or incubations should run under sterile conditions to minimize cells’ exposure to an unprotected environment. More exotic robotic plate handlers incorporate a HEPA filter to keep contaminants away from cells. High-level biosafety systems employ an enclosure to isolate workers and the lab environment from the assay.
Yet no microplate handler/reader system reliably duplicates the tight environmental control of a carbon dioxide incubator. Approximations are possible—for example, Tecan’s MIO (monitored incubator option), available with or without shaking options to ensure proper mixing of detection reagents.
Tecan has partnered with best-in-class vendors to provide cell assay workstations incorporating environmental control technologies that do not fall within the company’s core competencies. One of its successes was to extend the workstation idea beyond HPLC, extraction, and sample preparation to cell-based assays. This platform approach is ideal for organizations running the same types of assay day in and day out, such as a pharmaceutical toxicology lab.
Yet these systems also provide user-configurable flexibility and versatility. Tecan’s top systems for cell-based assays are, according to Braum, easily repurposed for other assay types. “We’ve used similar configuration for cell-based and biochemical assays,” she says, where differences might encompass reader settings or swapping in a multimode reader with luminescence, fluorescence, and absorbance. Tecan’s customers, she continues, rarely run just one type of assay. Braum explains, “They use these systems for a variety of assays and need built-in flexibility.”
For additional resources on microplates for cell-based assays, including useful articles and a list of manufacturers, visit www.labmanager.com/microplates