Guidelines for the Provision of Assay Buffer Residual Volumes in Microplate Wash Assays
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Guidelines for the Provision of Assay Buffer Residual Volumes in Microplate Wash Assays
Authored by: Peter Brescia, Applications Scientist and Peter Banks, Scientific Director, BioTek Instruments, Inc. Winooski, VT
Setting aspiration manifold Z-heights to provide predetermined residual volumes in the final dispense/aspirate step of a microplate wash cycle
Contents |
Introduction
Z-height of the aspiration manifold can be used to leave behind accurate residual volumes of assay buffer in the final dispense/aspirate step of a wash cycle. The properties of the assay buffers, such as ionic strength and the use of detergents, can have a significant impact on the residual volumes.
Wash assays are indispensable formats for the selective and sensitive quantification of countless analytes. Wash steps provide the means for removal of interfering components in the sample matrix and excess added reagent.
Many applications require that a predetermined residual volume be left in each microplate well following the final dispense/aspiration step of the wash cycle. Figure 1 details an immunocytometric assay to determine the activation and translocation of glucokinase in rat primary hepatocytes, and involves numerous steps requiring different residual volumes.[1]
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| Figure 1. Workflow for gluocokinase translocation assay in microplate format showing assay component addition (green); washer operations (red); incubation conditions (black); and imaging reader (IN Cell 3000 Analyzer, GE Healthcare) (blue). |
Several factors can affect the assay buffer volume remaining after the last wash cycle aspirate/dispense step in addition to obvious parameters such as aspiration manifold Z-height and microplate well density/volume. These include ionic strength of the assay buffer and various additives (ex. detergents), microplate composition (polystyrene, polypropylene, etc.) and any microplate coatings that can affect fluid surface tension.
In this study, we provide guidelines for the provision of residual volumes based on aspiration manifold Z-height for assay buffers differing in ionic strength. Additionally, the effect of Tween 20 for Corning 96-well (untreated and tissue culture-treated) and 384-well (untreated) densities is examined. Finally, some common buffers used for cell-based assays, including Tyrode’s buffer and DMEM-F12, will be tested. Testing was performed with the EL406 Microplate Washer Dispenser (Figure 2) from BioTek Instruments (Winooski, VT).
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| Figure 2. Microplate washer dispenser used to provide predetermined residual volumes. |
Methods
For the residual volume testing described below, Liquid Handling Control™ (LHC) Software from BioTek Instruments was used to program the wash protocol. This option is defined in the wash step by selecting Show Wash Options (Figure 3a).
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| Figure 3. Programmed wash protocol options. |
Once selected, the screen will expand to include Final Aspirate Definition (Figure 3b). By checking the options box and clicking on Definition, the final aspiration parameters can be set independently of other aspiration steps in a multi-step wash protocol. Here one can define the Travel Rate between settings 1-5 for non-cell based assays or 1-4CW or 6CW for cell-based assays (CW) and delay time at bottom of well during the aspirate step (Figure 3c). To define the Z, X and Y positions, select Advanced options (Figure 3d).
The positions are defined in motor steps, although values entered are immediately converted to units of measure (mm). Therefore, it is possible to adjust these values in regards to well position if the plate geometry is known. Furthermore, by calculating the cylindrical volume, one can estimate a starting point for the Z-height position (Figure 3e) remembering that the well bottom may be up to several millimeters above the carrier.
The experimental values shown in Figures 4-8 were determined by making incremental changes to the Z-height offset to help determine a representative value range for a sample of plate formats and types. Due to the diverse needs of various experimental protocols, each solution/plate combination will require additional empirical testing to determine the optimal settings for a particular residual volume.
Residual volume testing was performed to determine the average residual volume per well, and standard deviation was expressed in units of volume (μL) at a 95% confidence interval. All measurements included performing the wash protocol in triplicate for each plate type at each Z-height offset indicated in Tables 4-8. The average residual volume per well was calculated using the residual volume mass and solution’s density. Each solution included FD&C #1 blue dye to obtain a final OD 450 nm between 0.1 and 1.0 using dual-wavelength measurement (630 nm - 450 nm).
Residual volume per well was calculated based on the absorbance measurement data using a residual factor (mean OD450/avg mass per well) * OD. Residual volume per well was then used to calculate standard deviation and 95% confidence intervals (Microsoft® Excel® spreadsheet software).
Results and Discussion
Corning, Flat Bottom 96-well Microplate (p/n 9017)
For 96-well microplates, aspiration manifold Z-heights reflecting residual volumes in the range of 25-100 μL (~8% < well volume < 33%) were investigated. Volumes lower than 25 μL were not investigated as these tended to provide incomplete coverage of well bottom surface due to surface tension.
Effect of Buffer Concentration
The effect of buffer concentration on residual volumes is demonstrated in Figure 4. It is apparent that there is a significant difference in residual volume obtained for like Z-height settings when HEPES buffer concentration is increased to 100 mM.
For a given Z-height, there is approximately 20 μL less residual volume left behind using 100 mM HEPES relative to 10 mM HEPES or de-ionized water. At a 95% confidence level, there is no difference in residual volumes for a given Z-height between 10 mM HEPES and de-ionized water.
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| Figure 4. HEPES buffer concentration effect on residual volumes as a function of aspiration manifold Z-height. |
Effect of Detergent – Tween 20
Detergents act to lower fluid surface tension, which can impact Z-height control of residual volumes. Figure 5 demonstrates the effect of 0.1% and 1.0% Tween 20 on residual volumes as a function of aspiration manifold Z-height.
At a 95% confidence level, there is no difference between the use of de-ionized water, 0.1% Tween 20 and 1.0% Tween 20.
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| Figure 5. Tween 20 concentration effect on residual volumes as a function of aspiration manifold Z-height. |
Use of Common Cell-based Assay Buffers
Cell-based assays often use media such as DMEM-F12, which contains amino acids and glucose as well as salts to buffer and create an isotonic environment for the cells. DMEM is typically used while plating cells and can also be used during cell stimulation when receptor agonists are added in conjunction. Addition of detection reagents often uses a common buffer such as Tyrode’s buffer. While the buffer is devoid of amino acids, the solution is isotonic with cells and does usually contain glucose. Figure 6 portrays residual volumes as a function of aspiration manifold Z-height for these two common buffers in comparison to de-ionized water.
Surprisingly, there is no significant difference in residual volume for a given Z-height between these high ionic strength assay buffers and de-ionized water at the 95% confidence level. Presumably, there are parameters other than ionic strength at play here that make for different performance relative to 100 mM HEPES. For example, Tyrode’s buffer contains 1% bovine serum albumin, which can influence the residual volume and Z-height relationship.
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| Figure 6. Effect of common cell-based assay buffers on residual volumes as a function of aspiration manifold Z-height. |
Effect of Microplates Treated for Tissue Culture
Often, cell-based assays use polystyrene microplates that have been specially treated for sterility and good cell adherence. The tissue culture treatment cross-links carboxyl and amine groups with the correct net charge, and is typically applied in a plasma oven. This attracts mammalian cells and promotes good adherence necessary for satisfactory assay performance. Figure 7 examines the effect of tissue culture treatment on a 96-well flat bottomed microplate using Corning’s tissue culture-treated plate (p/n 3603) and the HEPES buffers.
It is apparent that tissue culture-treatment of polystyrene flat-bottomed plates negates the effects of buffer concentration seen earlier. From data available in the appendices, all fluids and buffers tested provided equivalent residual volumes for a given aspiration manifold Z-height. This suggests that tissue culture-treated microplates should be used for accurate provision of residual volumes without influence from assay buffer additives.
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| Figure 7. Tissue culture-treated microplate effect on residual volumes as a function of aspiration manifold Z-height. |
Corning, Flat Bottom, 384-well Microplate (p/n 3702)
Aspiration manifold Z-heights in 384-well microplates were investigated that provided residual volumes ranging from 5-25μL (~6% < well volume < 30%). It is suspected that the reduced surface area of the 384-well plate compared to the aspirate pin size may negate any solution effects seen with the untreated 96-well microplate. Thus, experiments were performed with an untreated microplate. Figure 8 demonstrates a lack of statistically relevant effect on residual volumes as a function of aspiration manifold Z-height for increasing buffer concentrations or use of detergent.
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| Figure 8. HEPES buffer concentration effect on residual volumes as a function of aspiration manifold Z-height. |
Conclusions
Tissue culture-treated microplates appear to be the best option for the most accurate provision of residual volumes. This is a fortunate circumstance as most applications requiring residual volumes at the end of a wash cycle involve cell-based assays, which are particularly suited to tissue culture-treated plates. Table 1 provides some guidelines for the selection of aspiration manifold Z-height to provide certain residual volumes in both 96- and 384-well Corning, flat bottomed, tissue culture-treated microplates. In the case of 384-well microplates, tissue culture-treated microplates are not required to reduce buffer effects on residual volumes, but for cell-based assays, it is still recommended to use the tissue culture-treated options.
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| Table 1. Guidelines for residual volumes in tissue culture-treated plates based on aspiration manifold Z-height settings. Note: settings may vary somewhat depending on microplate manufacturer and the use of assay buffers not tested here. |
References
- ↑ M. Wolff et al. (2008) Journal of Biomolecular Screening, 13(9), pp. 837-846.
Appendices – Original Data Tables
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| Table A1. Corning Costar 96-well flat-bottom plate used to measure residual volume at various Z-axis manifold positions on an ELx405. Rows without data indicate that residual volume was not measured at that Z-axis manifold position. |
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| Table A2. Corning Costar 96-well flat-bottom tissue culture treated plate was used to measure residual volume at various Z-axis manifold positions on an ELx405. Rows without data indicate that residual volume was not measured at that Z-axis manifold position. |
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| Table A3. Corning Costar 384-well flat-bottom plate Tissue Culture treated was used to measure residual volume at various Z-axis manifold positions on an ELx405. Rows without data indicate that residual volume was not measured at that Z-axis manifold position. |
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