A Focus on Liquid Handling

Best practices for not disrupting cell monolayers:

When used correctly, automating liquid handling steps for cell-based assay can increase throughput and reliability. However, the use of fluid dispensing or aspiration steps that are inappropriate for the conditions (e.g., a dispense rate that is too rapid) can cause a portion of the cell layer to be either washed off or folded over onto adjacent cells. The 406 FX washer dispenser fluidics are designed to allow for slow fluid rates, to avoid these potential problems while retaining dispense accuracy. The dispense tips are also angled vertically, to dispense toward the side of the well rather than directly onto the cell monolayer.

An additional important consideration is the column of fluid already present in the well, which can help prevent the disruption of cells in the well. The fluid column serves as a mechanical “shield” between the fluid jet delivered to the well and the cells on the bottom. The greater the fluid column (i.e., larger the volume), the more effective the shielding against fluid-induced cell damage. The aspiration height parameter can be used to modulate the fluid column between wash cycles. By using a height parameter that keeps the aspiration tube off the bottom of the well, residual fluid can intentionally be left in the wells. The elevated aspiration height serves a second purpose—positioning the aspiration tubes close to the cell layer for an extended period is harmful to the cell monolayer. A small yet strong vortex occurs near the aspiration tube that can damage fixed or live cells. The higher aspiration height increases the distance between the tube and the cell monolayer and provides some protection. Also, the 406 FX is configured with programmable aspiration rates that enable very rapid aspiration dive rates that minimize the exposure of cells to this deleterious force.

Optimizing settings for ELISA assays:

BioTek Agilent washer dispensers such as the 406 FX have preloaded default parameters that will readily perform the wash for most ELISA assays. However, differences in plate geometries, assay subtleties, and different buffer constituents may necessitate that some parameters be optimized for the best washing performance. The 406 FX has several different parameters that can be varied for both aspiration and dispense. For example, the horizontal position and vertical height of the dispense tubes can be altered. The manifold has angled dispense tubes, and by changing the dispense X-axis parameter, the dispense tube can be shifted toward one side of the well. Different plate heights can be accommodated by altering the Z-axis setting.

Similar parameters are available for the aspiration tubes as well. While the default position is the center of the well, several plate types require aspiration from the well edge for more effective aspiration. Likewise, differences in the physical height of the microplate bottom require a different aspiration Z-height setting. To minimize the buffer residual, the aspiration tubes need to be as close to the bottom as possible, without touching the well. The aspiration rate can also be changed from very gentle to aggressive, depending on the nature of the assay.

Once the wash parameters have been optimized, there are two critical items that need to be addressed. First, it is necessary for the wash programming to follow the assay kit instructions. It is critical that the correct number of wash cycles, dispense volume, soak time, and shaking (if required) are correctly programmed. The wash parameter instructions for assay kits have been optimized by the kit manufacturer. Immediately before the run, it is important to confirm that the washer supply and waste bottles have been attached correctly. While the supply container is not pressurized, the waste bottle is under vacuum and the top needs to be seated correctly and the tubing connected. Fluid flow sensors would alert the user, but a little bit of care can save time. Second, the washer must be primed with the appropriate buffer. This step removes residual fluids and air and replaces them with the appropriate buffer fluid. Failure to do so will result in either insufficient wash buffer or a dispense with an incorrect solution.

Visual Abstracts

Visual abstracts provide an animated, visual representation of the assay.

• Automated Cell Fixing, Staining, and Immunofluorescence in Microplates
• Automated Reagent Addition and Washing of ELISpot Microplates
• Automated Reagent Addition and Washing of Magnetic Bead Assays
• Automated ELISA Liquid Handing with the Agilent BioTek 406 FX Washer Dispenser
• Automation of Cell-Based Drug-Absorption Assays
• Automated Washing and Reagent Dispensing for Cell-Based Assays With Verification of Cell Retention