Cell Deposition and Drug Sensitivity Assay Protocol

This protocol describes how to deposit several cells of S. robusta in droplets on a petri dish, allowing the experimenter to track the growth of approximately single-cell isolates with high throughput. This was used to determine drug sensitivity. We found that in, liquid culture, S. robusta is sensitive to 10 μg/mL puromycin and 250 μg/mL glyphosate. One caveat to remember is that S. robusta appears more tolerant to antibiotics when grown on agar plates, insensitive to up to 20 μg/mL of puromycin, for example. We recommend selection in liquid culture.

Measure the density of floating cells in the culture

• Grow 100 mL volume of cells in a 175 cm² ask for three to four days, until there is a substantial number of healthy, floating cells.
• Stand to ask vertically and take a 15 mL volume of the floating culture (not to disturb the adherent cells) to measure cell density.
• Spin cells down at 6000xg to pellet them. Scrape and resuspend in 1 mL.
• Measure cell density using a hemocytometer. The target concentration for distributing cells is 400-500 floating cells/mL, so dilute as necessary. The volume needed is 2 mL, including any drugs you want to add.

Deposit cells in droplets on a petri dish

• Take the 2 mL, containing the desired number of cells, in an Eppendorf tube.
• Centrifuge using a personal mini-centrifuge for two minutes. At this speed (which I believe is 6000 rpm), many cells remain in the supernatant. Those are the cells we want. Do not disturb any pellet that forms from the centrifugation.
• Using a Pipetteman, withdraw 40 μl aliquots from near the top of the Eppendorf tube. Deposit 40 μl drops in a 6x8 array in a Petri dish.

Count initial cells

• Record the approximate number of cells deposited in each drop.
• In our hands, this is 4-10 cells per droplet on average.
• Wrap petri dish with parafilm and place in incubator.

Assay

• After three days of treatment, count cell numbers/determine cell viability in each droplet.

Creative Bioarray Relevant Recommendations

• Drug-resistant cell models are developed in our laboratory by pulsing or repeatedly exposing cancer cells growing in cell culture to drugs. Creative Bioarray chooses suitable selection strategies such as pulsed selection strategy or continuous selection strategy based on the customer's requirements on cell type, chemotherapy agents, resistance level, etc.
• We provide various in vitro ADME/PK services, including high-throughput ADME screening, in vitro binding, in vitro metabolism, in vitro permeability, and transporter assays. We also provide in vivo drug metabolism and pharmacokinetic (DMPK) services to support drug development studies of in vivo absorption, distribution, metabolism, and excretion of drug candidates. Our in vivo DMPK services cover a comprehensive range of different animal studies in several species.

• Standardize the method of cell deposition (e.g., seeding density, method of plating).
• Use appropriate vessels (e.g., 96-well plates, flasks) and ensure they are sterile.
• Prepare drugs in a consistent manner (stock solutions, dilutions).
• Use appropriate solvents and ensure they do not affect cell viability.
• Define the concentration range and exposure times for drug treatments.
• Include positive and negative controls for comparison.

In Vitro DMPK Services

In Vivo DMPK Services