Cell and molecular biology become more integral pieces of the modern drug discovery process on a seemingly daily basis. Monitoring the interaction of cells and internal cellular activity provides vital information about cell health, changes in disease states, and the response of targets to potential therapeutic compounds.
Figure 1 – Work flow of automated microtiter plate-based cell staining process.
The ability to visualize biological activity at the cellular level has been a key element in drug discovery. The addition of automation and high-volume screening methods provided the ability to test large numbers of compounds. Together, imaging and screening are significant technological drivers of pharmaceutical research.
Figure 2 – Automated cell staining method on the Biomek 4000 Laboratory Automation Workstation. a) Screen shot of the Define Pattern step in the automation method. Users can freely choose the sample wells on a 96-well plate format. b) Screen shot showing the deck layout. Tools, labware, and tips are located as indicated. c) Screen shot of User Interface, where users can customize all the parameters, including reagent volumes, washes, incubation times, and antibody information. d) Screen shot showing the reagent volumes based on the parameters from User Interface. e) Screen shot showing the custom-defined antibody transfer step, where users can define the antibody locations manually.
Cancer cells offer an illustrative example. Screening large volumes of samples, usually in microplates on robotic platforms, enables efficient testing of a compound against a specific type of cancer at various stages in its development. Alternately, researchers can monitor the cellular changes that occur in response to a compound over time.
In genetic research, expressed proteins provide important indicators of successful up-regulation or down-regulation of proteins and activities governed by newly introduced genes. Again, changes in cellular behavior over time can be valuable indicators of biological functionality, response levels, and other beneficial indicators of treatment viability.
Cell staining is a very time- and labor-intensive process, alternating between pipetting steps and incubation periods, and can take hours or even a day with large batches. While important time-savings are achieved, so, too, is consistency, from lot to lot, batch to batch, and day to day. These benefits hold true throughout the drug discovery process and with all kinds of cells.
The following application illustrates the cell staining work flow on an automated platform and details the results and the benefits that accrue.
Figure 3 – Intracellular staining of stem cell markers using PerFix-nc reagents. PerFix-nc fixation and permeabilization reagents are optimized for no-wash detection of intracellular markers for flow cytometry, but also provide excellent sample preparation for imaging applications. The PerFix-nc kit was tested on undifferentiated and day 8 differentiated mES cells for staining. Mixtures of mES cells and day 8 differentiated mES cells (ratio 1:2) were detached and reseeded into a 96-well imaging plate, then allowed 24 hr to plate out. PerFix-nc reagents and antibodies were added into the sample wells. All liquid handling steps were performed on a Biomek 4000 platform. Sixteen wells required less than 1 hr to prepare for imaging. Images were taken using the 10× objective on an ImageXpress high-content imager and processed with MetaXpress software (Molecular Devices, Inc., Sunnyvale, CA). Cells were stained with either isotype controls (B.a.) or with myosin heavy chain (green, A.b.) and Sox2 (blue, A.c.). DAPI (red, A.d) was used to mark the locations of nuclei. The group treated with cardiomyocyte inducing reagents (B.c.) has more MF20 positive staining than the nontreated group (B.b.).
Materials and methods
Culture and differentiation of mouse embryonic stem cells
Mouse embryonic stem (mES) cells (Invitrogen, Carlsbad, CA) were maintained in growth media containing leukemia inhibitory factor (LIF) and 15% knockout serum replacement (KSR). For differentiation, cells were cultured in 15% fetal bovine serum (FBS) without LIF in a 384-well round-bottom polypropylene plate (Nunc, Roskilde, Denmark) in 40 μL differentiation medium (various treatments for 0–5 days). Embryoid bodies formed by day 5 were transferred to gelatin-coated 96-well plates in 100 μL fresh media. After day 6, a portion of the adherent cells showed visible contraction. Control mES cells were treated in parallel without differentiation factors. Cells were harvested by trypsin and dispensed into 96-well plates for processing.
Figure 4 – Mixtures of mES cells and feeder cells were seeded into a 96-well imaging plate, then allowed 24 hr to plate out before being treated with PerFix-nc reagents and stained with either anti-Nanog-Alexa Fluor 488 (b, green) or anti-Sox2-Alexa Fluor 647 (c, blue). DAPI (d, red) was used to mark the locations of nuclei.
mES cells and cardiomyocyte characterization
Differentiated cells were harvested on day 8 using Accumax cell detachment solution (Millipore, Billerica, MA). Then, mES cells were mixed with either pooled differentiated cells or feeder cells (Invitrogen), and seeded into 96-well imaging plates and allowed 24 hr to attach. Using a Biomek 4000 Laboratory Automation Workstation (Beckman Coulter Life Sciences, Indianapolis, IN) with an eight-channel pipetting tool (Figure 1), the mixed cells were fixed and permeabilized with PerFix-nc reagents (Beckman Coulter) using the Biomek Cell Staining automation method (Figure 2). The mixture of differentiated cells and mES cells were stained with myosin heavy chain-Alexa Fluor 488 (clone MF20, eBioScience, San Diego, CA) and anti-Sox2-Alexa Fluor 647 (clone O30-678, BD Biosciences, San Jose, CA) (Figure 3). The mixture of mES cells and feeder cells were stained with either anti-Nanog-Alexa Fluor 488 (clone eBioMLC-51, eBioScience) or anti-Sox2-Alexa Fluor 647 (clone O30-678, BD Biosciences). All conjugates were titered for optimal performance, and relevant isotype controls were used to control for nonspecific staining. Cells were fixed by adding 5 μL of PerFix-nc reagent one for 15 min, followed by permeabilization and staining with 50 μL of PerFix-nc reagent two containing the antibody conjugates for 30 min. After removal of the supernatant and replacement with 50 μL reagent three for 5 min, cells were identified by nuclear staining with mounting medium containing 4’,6-diamidino-2-phenylindole (DAPI) (Invitrogen).
All samples were analyzed on an ImagXpress system with MetaXpress software.
Expression of myosin heavy chain in differentiated cardiomyocytes (Figure 3) and stem cell markers (Figure 4 and Table 1) were clearly demonstrated in the cell preparation processed with the Biomek 4000 Laboratory Automation Workstation. Automated cell staining for 58 wells on the liquid handler required about 1 hr without user intervention. The PerFix-nc sample preparation system provided good intracellular staining results with reproducibility and accuracy without washing, greatly facilitating assay automation.
This work demonstrates that cell staining sample preparation work flows can be automated using standard components on the Biomek 4000 Laboratory Automation Workstation. Automation can achieve preparation time savings with large numbers of samples while maintaining equivalent results and precision compared with manual processing. As a result, it is possible to perform large cell staining studies in an expandable manner with walkaway capability. Furthermore, all steps are optional and the system will readily adapt to individual work flows.
Amy Yoder and Li Liu, Ph.D., are Senior Development Scientists, Beckman Coulter Life Sciences, 5350 Lakeview Parkway Dr. South, Indianapolis, IN 46268, U.S.A.; tel.: 318-808-4200; e-mail: firstname.lastname@example.org and email@example.com. Note: Beckman Coulter, the stylized logo, and Biomek are trademarks of Beckman Coulter, Inc., and are registered in the USPTO. All other trademarks, service marks, products, or services are trademarks or registered trademarks of their respective holders.