*3.3.1. Volume variation*

The volume delivered by a pin can change due to a number of factors. To minimize volume variations, there should be consistency in immersion depth (Dunn and Feygin 2000). There is a minimum volume required in the source plate, and the destination plate should not be dry (Rudnicki and Johnston 2009). The dwell time that pins spend in the fluid and with‐ drawal speed from the liquid surface should be optimized for solutions of very different properties (e.g. viscosity).

The slot of a pin can be tainted by compound precipitation or formation of suspension de‐ posits (Fig. 3B). Sufficient and robust washing and drying steps are effective in preventing deposition and being critical to avoid carry-over and cross-contamination. The pins can be physically damaged by dipping in highly uneven surfaces, particularly when using slotted pins (Fig. 3C). Coated pins should avoid harsh washing procedures, such as going through powerful sonication washes.

*3.3.2. Carry-over*

fer time.

After transferring compounds from one plate to another, the pins are washed in DMSO, al‐ cohol, water or a combination of these solutions. The pintool protocol involves dipping the pins in each solution bath certain number of times, at a particular speed and soaking time. The pins are then dried on lint-free blotting paper. Protocols of pintool devices used on ro‐ botic platforms are optimized for effectiveness in removing previous transfers while spend‐ ing the minimum time between wash cycles. In many cases, the drugging (i.e., addition of compound to assay well) step using pintool becomes the bottleneck in a screening cam‐ paign, and the washing step accounts for most of the time consumed. However, certain as‐ says can be very sensitive to compound carry-over, particularly if the compounds are very potent modulators and bind avidly to the pin surface. In such cases, increasing the number of dips and soaking time can improve cleanliness, albeit at the cost of increasing total trans‐

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Fig. 4 illustrates the effect of four different wash protocols in a kinase assay using stauro‐ sporine as the inhibitor. After compound transfer by pintool to the first assay plate, the pins are immersed in DMSO and isopropanol reservoirs, followed by drying on blotting paper. Subsequently, the pins are dipped in a second assay plate containing the kinase system. Re‐ sidual staurosporine in the pins increases the signal variation as determined by %CV of a set of multiple wells. Protocol 1 has the least number of dips and soaking time per bath, result‐ ing in the most dramatic signal variation due to carry-over. This general approach is recom‐

**Figure 4.** General approach to detect compound carry-over and optimize pintool washing. A single wash cycle con‐

Regular pintool calibration and quality assessment can considerably improve data quality. In screening runs at a single compound concentration, well-maintained pins can lead to a

sists of dipping the pins in DMSO and isopropanol baths, followed by blotting on lint-free paper.

*3.3.3. Routine quality assessment*

mended for detecting carry-over and selecting the appropriate pintool wash.

**Figure 2.** A-B) Delivery variation by a bulk reagent dispenser distributing a FITC solution into 384-well plates. Certain dispensing cassette channels display either higher %CV or %bias' values than the anticipated cut-off of 10%. C) Cell settling in the reagent reservoir when transferring to a microtiter plate using an automated pipetting system with an 8-channel head, with 1 min delay between transfers to each column. Cell settling is uneven due to the v-shaped bot‐ tom of the reservoir, causing the intensity pattern observed in the plate. The cells (HEK293T) were incubated with Cell‐ Titer-Glo® for 20 min prior to luminescence reading.

**Figure 3.** Magnified view of FP1NS50H pins (V&P Scientific, Inc.) with A) clean slot B) dirty slot C) damaged slot.
