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White Paper by Duong Chau V&P Scientific Staff Scientist Abstract V&P Scientific VP 177AD-1 and VP 179BJD are dispensing manifolds designed for rapid filling of 96 and 394 well microplates respectively. This experiment was set up to determine both the accuracy and precision of these instruments by measuring the quantity of aqueous FITC dispensed per well and calculating the coefficient of variation among the wells and also the variation among the dispenses. The manifolds were tested in volumes of 50, 100, and 200 uL per well for the VP 177AD-1 and 25, 50, and 75 uL per well for the VP 179BJD. The coefficient of variations ranged between 3% and 7% using the VP 177AD-1 and between 4% and 5% using the VP 179BJD when dispensing occurred below the meniscus in contact with the liquid. Non-contact dispensing was examined using the VP 179BJD and resulted in coefficient of variations ranging between 6% and 9%. The VP 177AD-1 and VP 179BJD manifolds are capable of rapid dispensing into microplates. These instruments can instantly fill a microplate with one dispense, while maintaining acceptable precision. Standard pipettors often require multiple dispenses to fill a microplate, twelve dispenses for a 96 well microplate and thirty-two dispenses for a 384 well microplate using a 12-channel pipettor. This makes the VP 177AD-1 and VP 179BJD a convenient alternative to regular pipetting, or an economical replacement for expensive workstations.
Introduction Many high-throughput laboratories use microplates for their assays that often require some type of media or reagent buffer solution in addition to the compound being tested. Filling up a 96 or 384 well microplate requires the assistance of a liquid handling robotic station or a lab technician using a 12-channel pipettor. A robotic workstation is very expensive, oftentimes outside the budget of a small lab, while manually pipetting of a microplate is very time consuming. Even in larger labs, it is more efficient to have the workstation run assays rather than fill plates. V&P Scientific, Inc. has developed a series of manifolds which can dispense liquids in 96 and 384 well microplates. This report will focus on the VP 177AD-1 and VP 179BJD, a 96 well microplate and a 384 well microplate dispenser, respectively. With an experienced technician, microplates can be filled within 20 seconds while being easier to setup than a robot. These manifolds are designed for high-throughput labs with an economical budget in mind. The VP 177AD-1 is a dispensing manifold designed for rapid filling of 96 well plates. The manifold has 96 stainless steel pins protruding into the reagent chamber spaced 9 mm apart for a standard 96 well microplate. Plates can be filled with reagents by sliding plates under the manifold into the microplate indexer, adjusting the Z-Height, and using a liquid dispenser, such as a bottle top dispenser or syringe, to pump liquid through the manifold and into the microplate. The VP 179BJD applies the same conceptual design as the VP 177AD-1, but with 384 stainless steel tubes spaced 4.5 mm apart for 384 format microplates. Also, the VP 179BJD is mounted onto a VP 179A, allowing for precise indexing of a microplate, a necessary feature for 384 format microplates. This experiment was set up to determine both the accuracy and precision of these instruments by measuring the quantity of aqueous FITC dispensed per well and calculating the coefficient of variation among the wells and also the variation among the dispenses. Background Information The method of testing the VP 177AD-1 and VP 179BJD are fluorescence-based readings, using fluorescein isothiocyanate (FITC). FITC is a derivative of fluorescein with the molecule functionalized with isothiocyanate, thus making it reactive towards amine and sulfhydrl groups, and a great tool for labeling proteins. FITC has an excitation and emission wavelengths of approximately 495 nm/521 nm, and a maximum energy output at pH. 8.0. Because FITC is pH sensitive, it is important to keep it in a buffer to avoid even small shifts in pH1. The excitation value output of FITC is directly proportional to the amount of FITC molecules, hence a linear relationship between the amount of FITC and the excitation signal. Using this model, the experiment is designed such that the amount of FITC dispensed by the manifolds in each well is read, thus each well can be compared to one another. The percent coefficient of variation (%CV) can be extrapolated from this data using the formula:
The percent coefficient of variation is a recognized value in the laboratory for determining precision of instruments, thus allowing for comparisons to other equipments with similar functionalities (i.e. comparing the manifolds to multi-channel pipettors commonly found in laboratories)2. Materials and Methods
· Dimethyl Sulfoxide (Sigma D2650) · Tris-HCl, 1 M Stock Solution, pH 8.0 (Sigma T3038) 100 milligrams of FITC was dissolved in 4 mL of DMSO and left overnight to ensure complete dissolution and equilibration of the mixture. Mix 80 uL of the FITC mixture with 2000 mL Tris-HCl, 0.1M, pH 8.0, buffer to get a final concentration of 0.001 mg/mL FITC solution. This is the solution to be dispensed during the assay. The final concentration to be used is a function of several factors, the most important to consider are the following; the readable linear range of the plate reader, the strength of the fluorescence, and the volume dispensed into the wells. A standard curve was generated using a 12-channel pipette to determine the ideal concentrations and volume of FITC to be used, hence the concentrations used here reflects the equipment and reagents used here. The manifolds were set up according the manufacture’s technote. The FITC solution was dispensed into the microplates using the VP 177AD-1 at dispense volumes of 50, 100, and 200 uL, and volumes of 25, 50, and 75 using the VP 179BJD. These transfers were done below the meniscus such that the liquid would wick off any remaining drops on stainless steel tubes (contact dispense). Another dispense technique was examined with the VP 179BJD where the stainless steel tubes remained above the wells when dispensing (non-contact), at dispense volumes of 25, 50 and 75 uL. These conditions were tested in triplicates (replicates of three).
Results Table 1 represents the data obtained from the VP 177AD-1 and VP 179BJD. It includes the CVs for each individual replicate as well as the average CV for the triplicate. Average CVs were 3.40%, 4.23%, and 6.40% for the 200, 100, and 50 uL transfers respectively using the VP 177AD-1. Average CVs for the VP 179BJD were 3.87%, 4.16%, and 4.18% for the 75, 50, and 25 uL transfers respectively. Non-contact dispenses using the VP 179BJD resulted in CVs of 6.67%, 7.46%, and 8.43% for dispense volumes of 75, 50, and 25 uL respectively. Discussion When using a common laboratory pipettor, oftentimes a droplet on the pipette is left behind and must be wiped off, thus a technician’s technique is imperative when attempting to achieve acceptable CVs. The same effect occurs in pipetting workstations as well, hence most of these workstations include a “blowout” step using air to dislodge any hanging droplets. When using V&P Scientific’s manifolds for non-contact dispensing, some droplets are left hanging while others fall into the wells. This seemingly random event from tube to tube results in higher CVs, compared to the contact dispensing (approximately a twofold increase). These increases in CVs can be explained by the wicking effect attributed to contact dispensing but absent in non-contact dispensing. As the volume dispensed decreased, CVs increased, a pattern observed for all different types of transfers. This effect was expected as with all instruments of this nature; as desired volume dispense reduces, it becomes more difficult to maintain low CVs. However, the VP 177AD-1 and VP 179BJD were capable of maintaining acceptable (below 10%) CVs for transfers as low as 50 and 25 uL per well respectively. Conclusion The microplate is essential to high throughput screening, hence many companies are dedicated to the development of instrumentations for dispensing into microplates. The “low-tech” end of the spectrum includes the multi-channel pipettors, which can reliably fill microplates, but can be time consuming and requires an experienced technician. The “high-tech” instruments such as the pipetting workstations can also reliably fill microplates, however, these devices are very expensive, oftentimes out of the budget of small labs. Even large labs will find that their workstations are often better utilized for assays rather than filling plates with buffers. The VP 177AD-1 and VP 179BJD manifolds are capable of rapid dispensing into microplates. These instruments can instantly fill a microplate with one dispense, while maintaining the precision of standard multi-channel pipettors. This makes the VP 177AD-1 and VP 179BJD an efficient and convenient alternative to regular pipetting, or an economical replacement for expensive workstations. References
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