*3.5.2. Washer performance*

Although compatibility and control properties are important, plate washers are predomi‐ nantly evaluated by their wash performance. Plate washers provide a range of user-defined dispense/aspirate heights, flow rates, and needle probe positioning in reference to the well walls. By adjusting these parameters for each step of the wash cycle, optimal wash perform‐ ance can be ensured. On the other hand, an adequate wash quality needs to be reached to diminish extensive background signal and high signal variations amongst wells. This can be primarily achieved by minimizing the amount of liquid left inside each well at the end of the aspiration step. Besides their effects on wash power, the above-mentioned parameters also have an impact on the residual volume and need to be fine-tuned in conjunction with the vacuum/pump settings. Some plate washers may also provide multipoint, secondary, crosswise or delayed aspiration modes aiming to deliver the best results. The number of wash cycles and the length of soaking time are other settings that can be modified to reduce back‐ ground noise levels.

#### *3.5.3. Washer maintenance*

Since plate washers consist of tubing and needles which transport buffer solutions or waste liquid to or from the device, they require special cleaning processes as they are prone to be clogged by chemical residues such as salt and proteins from the wash liquids. Depending on the frequency of use, the fluid path may need to be rinsed daily to prevent blockage and contamination, especially if different buffers are being delivered through the same tubing. An efficient cleaning method alternates deionized water and a detergent such as Terg-a-Zyme®, which is highly recommended by plate washer manufacturers. Plate washers which provide an automatic cleaning feature or integrated ultrasonic washing technology are often easier to maintain. Models which do not contain built-in cleaning functionality are generally supplied with removable dispense/aspiration manifolds to ease the maintenance tasks. Cleaning of the other detachable or fixed plate washer components should also be per‐ formed periodically.

#### *3.5.4. Troubleshooting*

are designed to accommodate labware with dimensions conforming to ANSI/SBS standards,

Both the vacuum assembly and the bottle setup are also important aspects of the plate wash‐ er. Although most washers operate through changes in vacuum pressure, pump-based vac‐

Plate washers functioning by positive displacement principle are also available, enabling non-contact washing with no residual volume (Rudnicki and Johnston 2009). For assays where more than one wash buffer may need to be used, plate washers with multiple dis‐ pense channels and automatic buffer switching capability are preferred to minimize both operation time and contamination. Examples of other optional features for safe instrument operation include waste liquid level sensors and plate detection sensors to avoid unwanted overflows and jams. For BSL2 or higher level experiments, a washer with aerosol cover

Although compatibility and control properties are important, plate washers are predomi‐ nantly evaluated by their wash performance. Plate washers provide a range of user-defined dispense/aspirate heights, flow rates, and needle probe positioning in reference to the well walls. By adjusting these parameters for each step of the wash cycle, optimal wash perform‐ ance can be ensured. On the other hand, an adequate wash quality needs to be reached to diminish extensive background signal and high signal variations amongst wells. This can be primarily achieved by minimizing the amount of liquid left inside each well at the end of the aspiration step. Besides their effects on wash power, the above-mentioned parameters also have an impact on the residual volume and need to be fine-tuned in conjunction with the vacuum/pump settings. Some plate washers may also provide multipoint, secondary, crosswise or delayed aspiration modes aiming to deliver the best results. The number of wash cycles and the length of soaking time are other settings that can be modified to reduce back‐

Since plate washers consist of tubing and needles which transport buffer solutions or waste liquid to or from the device, they require special cleaning processes as they are prone to be clogged by chemical residues such as salt and proteins from the wash liquids. Depending on the frequency of use, the fluid path may need to be rinsed daily to prevent blockage and contamination, especially if different buffers are being delivered through the same tubing. An efficient cleaning method alternates deionized water and a detergent such as Terg-a-Zyme®, which is highly recommended by plate washer manufacturers. Plate washers which provide an automatic cleaning feature or integrated ultrasonic washing technology are often easier to maintain. Models which do not contain built-in cleaning functionality are generally supplied with removable dispense/aspiration manifolds to ease the maintenance tasks.

an ideal plate washer is also able to support flat, v-shaped and round-bottom plates.

uum-free and pressure-free systems are also offered.

should be chosen to prevent spread of the contagious material.

*3.5.2. Washer performance*

192 Drug Discovery

ground noise levels.

*3.5.3. Washer maintenance*

Plate washers serve as an excellent alternative to time consuming manual wash procedures for many applications. Since all the wash parameters should be optimized for each specific application during the assay development stage, a tedious troubleshooting process may be inevitable while setting up wash protocols to meet specific assay needs. Table 1 presents a summary of wash parameters/components and their contributions to the wash performance along with various troubleshooting tips. Different assay types may require distinct consider‐ ations. With biochemical assays, minimizing the background signal and well-to-well varia‐ tions are the most important tasks in the optimization process. Low background signal levels can be achieved by reducing the leftover liquid volume in each well. Decreasing the aspiration height and lowering the aspiration rate can greatly affect the residual volume leading to minimal liquid amounts in the wells. In order to prevent high standard devia‐ tions in the assay readouts, equal residual volumes should be attempted by optimizing the aspiration/dispense heights and rates. Depending on the viscosity of the wash buffer, high aspiration rates or low dispense rates may lead to unequal volumes. Inadequate priming volumes, unadjusted dispense or aspiration heights, clogged tubing, and physical misalign‐ ments between the manifolds and plate carrier should also be avoided to prevent high sig‐ nal variations. The effect of the aspiration height on the final residual volume is presented in Fig. 6 for both 96- and 384-well black plates with clear bottom. The volume of the residual liquid (water) per well was measured with the gravimetric technique at several selected as‐ piration heights on a Biotek EL405 microplate washer, while all the other wash parameters were kept constant. A rising trend is observed in the final volume as the aspiration height is increased.

**Figure 6.** Effects of aspiration height on residual volume. Residual volume was measured in a) 96-well and B) 384-well plates at various aspiration heights. Residual volume was increased as the aspiration height from the bottom of the well was increased.

In cell-based assays, gentle cell washing is one of the most critical factors to produce repro‐ ducible assay results, and it can be controlled by several settings such as aspiration and dis‐ pense rates, heights and horizontal positions. For loosely-adherent cells, the cell layer attached to the bottom of the well may be easily disrupted by rigorous wash cycles, and the aspiration and dispense rates should be set low enough to prevent turbulence inside the wells. For the same purpose, wash fluid should be dispensed at a distance from the well bot‐ tom and may be even be aimed at the well walls when possible. To observe the consequen‐ ces of inadequate washing and dispensing parameters on the cell layer endurance, a 3-cycle wash experiment was performed on HEK 293T cells, which are known for their low adher‐ ence and propensity to be frequently washed away in cell-based assays. The fixing solution was dispensed at medium speed, and the cells were washed before and after fixation. Repre‐ sentative images from wells containing an intact or damaged cell layer are presented in Fig. 7. When dealing with adherent cells, each step of the assay protocol should be optimized, including those involving other liquid handling devices such as bulk dispensers, pintools and pipettors.

**parameter/component effect troubleshooting tips**

residual volume, gentle/rigorous washing

gentle/rigorous washing

gentle/rigorous washing

gentle/rigorous washing

residual volume, aspiration/dispense performance

performance

performance

aspiration height residual volume,

dispense flow rate dispense volume,

dispense height dispense volume,

vacuum/pump assembly aspiration/dispense

plate carrier aspiration/dispense

**Table 1.** Wash parameters and troubleshooting advices

assay buffer properties

aspiration rate

prime dispense performance • prevent air bubble formation or no/uneven

horizontal aspirate position gentle/rigorous washing • prevent bead loss by offsetting the aspirate

horizontal dispense position gentle/rigorous washing • undisturbed cell layer if dispense position is offset

dispensing with adequate priming

Practical Considerations of Liquid Handling Devices in Drug Discovery

http://dx.doi.org/10.5772/52546

195

• higher residual volume if too fast • perturbed cell layer if too fast • uneven aspiration if too fast

• higher residual volume if too high • uneven aspiration if too low or too high

• undisturbed cell layer if high enough

position (for magnetic bead assays)

• uneven dispensing if too low or too high

• optimize for viscous/non-viscous buffer solutions • add surfactant to the buffer solution to reduce

• no/uneven aspiration with insufficient vacuum

• no/uneven aspiration or leakage if tubing is

• uneven aspiration/dispense if plate carrier is not

• plate is placed on the carrier with A1 in the correct

• enough plate clearance to prevent jams • higher throughput with lower plate clearance

• uneven dispensing if too slow • fluid overflow if too slow or too fast • perturbed cell layer if too fast • air bubble formation if too slow

• fluid overflow if too high

defective, bent or clogged

leveled or movement is blocked

to aim the well walls

surface tension

supply

position

well bottom

• perturbed cell layer if aspiration probes touch the

As with most high throughput instrument operations, it is a common practice to perform a periodic quality check on plate washers to assure a satisfactory wash performance at each use. It is important to perform these assessments with a wash buffer that has a similar vis‐ cosity to the buffers used in most of the applications. For evaluations on the residual vol‐ ume, one can perform a mock wash with a dummy plate and measure the leftover liquid volume inside the wells with a single or multichannel manual pipettor. For more accurate results, gravimetric or colorimetric techniques can be used to calculate the average volume per well. This way, one can also test if dispensing/aspiration is consistent in all the probes, and if there is any physical failure with any of the device components.


**Table 1.** Wash parameters and troubleshooting advices

In cell-based assays, gentle cell washing is one of the most critical factors to produce repro‐ ducible assay results, and it can be controlled by several settings such as aspiration and dis‐ pense rates, heights and horizontal positions. For loosely-adherent cells, the cell layer attached to the bottom of the well may be easily disrupted by rigorous wash cycles, and the aspiration and dispense rates should be set low enough to prevent turbulence inside the wells. For the same purpose, wash fluid should be dispensed at a distance from the well bot‐ tom and may be even be aimed at the well walls when possible. To observe the consequen‐ ces of inadequate washing and dispensing parameters on the cell layer endurance, a 3-cycle wash experiment was performed on HEK 293T cells, which are known for their low adher‐ ence and propensity to be frequently washed away in cell-based assays. The fixing solution was dispensed at medium speed, and the cells were washed before and after fixation. Repre‐ sentative images from wells containing an intact or damaged cell layer are presented in Fig. 7. When dealing with adherent cells, each step of the assay protocol should be optimized, including those involving other liquid handling devices such as bulk dispensers, pintools

**Figure 7.** Effects of non-optimized dispensing and washing on low-adherent cells. HEK293T cells were fixed, stained with Hoechst 33258 and imaged with Acumen eX3 in a 384-well black clear bottom plates. The fixing solution was dispensed by a Thermo Scientific Matrix® Wellmate®. Representative images (shown here in false color green) of A) an

As with most high throughput instrument operations, it is a common practice to perform a periodic quality check on plate washers to assure a satisfactory wash performance at each use. It is important to perform these assessments with a wash buffer that has a similar vis‐ cosity to the buffers used in most of the applications. For evaluations on the residual vol‐ ume, one can perform a mock wash with a dummy plate and measure the leftover liquid volume inside the wells with a single or multichannel manual pipettor. For more accurate results, gravimetric or colorimetric techniques can be used to calculate the average volume per well. This way, one can also test if dispensing/aspiration is consistent in all the probes,

intact cell layer and B) disrupted cell layers indicated cell loss due to harsh dispense and wash settings.

and if there is any physical failure with any of the device components.

and pipettors.

194 Drug Discovery
