Cell-Based Assay Preparation in 1536-well Microplates using BioTeks EL406 Microplate Washer Dispenser
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Cell-Based Assay Preparation in 1536-well Microplates using BioTek’s EL406™ Microplate Washer Dispenser
Authored by: Eric Sandberg, SRU Biosystems, Woburn, MA; Peter Banks, BioTek Instruments, Inc. Winooski, VT
Introduction
Label-free technologies are designed to monitor binding events without the need for reporter tags that can influence the binding kinetics and thermodynamics. Most common applications involve determining binding partner k-on and k-off rate constants and thus equilibrium dissociation constants, Kd, that define the binding strength. This is particularly useful in monoclonal HGH Advanced antibody clone selection, where low Kds (or high binding affinity clones) are desired.
Another popular application is cell-based assays for screening applications, where agonist binding to cell surface receptors (G protein-coupled receptors, receptor tyrosine kinases, ion channels, etc.) induces changes in cell morphology and/or adhesion that can be monitored by label-free technologies.
BIND® Technology Primer
The label-free BIND platform (SRU Biosystems, Woburn, MA) uses a novel optical effect (Figure 1) to provide sensitive cell adhesion measurement in proximity of the BIND biosensor surface. The biosensor incorporates a proprietary nano-structured optical grating adhered to the bottom of SBS-standard microplates of different well densities. The BIND unique hoodia biosensor grating reflects a narrow light wavelength range, termed the Peak Wavelength Value (PWV), which is measured with a spectrometer.
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| Figure 1. Optical path of BIND Reader. |
When a cell binds to the biosensor surface, the PWV increases. The difference in PWV is the BIND response that is output from the BIND reader. This response can be monitored kinetically, which is helpful in quantifying responses in myriad cell-based applications. Another PWV change can occur upon cell surface receptor agonist phen375 fat burner binding, which is attributable to signal transduction pathways inducing cell slim weight patch morphology changes that can be monitored by the biosensor. These BIND responses (Figure 2) can either be an increase or decrease in PWV.
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| Figure 2. BIND response is a differential signal associated with a binding event. |
The need for 1536-well capability in label-free, cell-based assays
The principal drivers for technology adoption in primary screening are cost followed by throughput. Assay costs per well must be low, typically around $0.10/well. As there are no reporter tags, the principal costs associated with a cell-based, label-free assay are cells and the biosensor microplate. The biosensor microplate cost is fixed and independent of well density, thus the assay cost per well can be reduced by more than an order of magnitude by using 1536-well densities relative to 96-well formats, and brings the BIND technology into the cost constraints of primary screening. This miniaturization also serves to reduce the number of cells needed in the screen to as low as 1,000 cells per well. This also opens up the option to use human primary cells, where supply is limited.
Cell-based assay preparation for 1536-well biosensor plates using EL406™
For good cell adhesion to the biosensor plate (especially for non-adherent cells), often a coating is applied to the biosensor’s titanium dioxide base. This is typically an inexpensive protein such as fibronectin, collagen or laminin. First, the biosensor plate is coated with a solution of the proteinaceous material, excess protein is removed through washing, cells are added, and the plate is gently washed to ensure that only the cells that adhere to the biosensor remain.
This application note will demonstrate the utility of using a combination microplate washer and dispenser to perform these assay preparation steps and provide some validation data in a label-free, cell-based assay of a GPCR target.
Materials and Methods
Protein extracellular matrix used to coat 1536-well biosensor microplates, and CHO-K1 and HEK-293 cell lines, are readily available from multiple vendors.
General Considerations for Cell Dispensing into 1536-well Densities
There are two different options for fluid dispensing. A peristaltic pump (peri-pump) dispenser uses autoclavable tubing cassettes to pump fluid from a reservoir to 96-, 384-, and 1536-well microplates. This dispenser has the capability to dispense as low as 1 μL to the full volume of a 1536-well plate in 1 μL increments. The peristaltic pump dispenser fluid path may be sterilized by autoclaving, therefore it is more useful for cell dispensing than syringe pumps.
On the other hand, syringe pump dispensers have a lower limit of 3 μL per well. These syringe pump dispensers can be used for cell dispensing when using liquid sterilization procedures such as >70% ethanol exposure.
A combination washer dispenser such as the EL406™ Microplate Washer Dispenser (Figure 3, BioTek Instruments, Winooski, VT) combines both peri-pump and syringe pump dispensing technologies in one unit along with plate washing, and will be used to demonstrate cell-based assay preparation.
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| Figure 3. EL406 Microplate Washer Dispenser. |
For 1536-well microplates, the 1 μL tubing cassettes used with the peri-pump dispenser are available with two different tip configurations. The sapphire-jeweled tip has a very small orifice for homogeneous solution dispensing, while the plastic molded tips have a larger opening and work better for cell suspensions, and they are less prone to clog with high-protein concentration solutions.
In regards to flow rates when using the two different 1 μL tip types, the sapphire-jeweled tips can be used at lower flow rates than the molded tips due to the smaller orifice and subsequently higher fluid exit velocities. In either case, it is important to use a rate sufficient to propel the fluid to the 1536-well microplate well bottom. As a rule, faster rates tend to work better. Both the jeweled and plastic tip 1 μL cassettes are optimal for dispensing from 1-50 μL. For larger volumes, 5 and 10 μL cassettes are also available.
General Considerations for Cell Washing in 1536-well Densities
The aspiration and dispense parameters used for 1536-well microplate washing are highly plate type- and process-specific. In regards to dispense height, manifold height adjustment such that it is less than 1 mm above the plate’s top is critical for faithful fluid dispensing. Likewise, the aspiration height is set based on its position Phen375 relative to the bottom of the well.
The microplate washer dispenser is controlled using an external PC that is running Liquid Handling Control™ (LHC™) Software. The fluid addition procedures can be accomplished using the peristaltic pump dispenser with a 1 μL cassette. Each well received 10 μL of fluid with the cassette set with a Z-axis position of 225 (10.29 mm above carrier) and both X- and Y- axis positions set to 0 (center of the well). The dispense rate should be set to “high” and the pre dispense-prime set for 2 dispenses of 10 μL/tube.
The aspiration of fluid during wash steps can be carried out using a travel rate setting of 6 CW (14.7 mm/sec) with no delay. The aspiration manifold can be positioned with a Z- axis setting of 10 (1.2 mm above carrier) and both X- and Y- axis positions set to 0 (center of the well).
Results and Discussion
Protein Extracellular Matrix Addition
A syringe pump on the EL406 was used to deliver microliter amounts of protein proactol plus solution suitable for providing a matrix layer on which cells could adhere to the biosensor microplate. Figure 4 demonstrates the significant PWV shift, 200 pm < PWV shift < 1000 pm depending on protein matrix, seen with the protein matrices relative to the uncoated biosensor microplate.
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| Figure 4. PWV shift and inter-plate reproducibility due to extracellular matrix (ECM) coating of biosensor microplates. |
The significant increase in PWV can be used as a QA/QC test to ensure appropriate protein performer 5 matrix coating of the biosensor microplate well is achieved. Good reproducibility in PWV shift relative to uncoated is seen between different biosensor microplates.
ECM coating across a plate is also an important consideration for reproducible cell-based assays. Intra-plate reproducibility was determined using the BIND SCREENER software and its ability to visualize clear skin max reproducibility with the aid of heat maps. Figure 5 demonstrates the reproducibility of ECM deposition across a 1536-well biosensor microplate for both EL406 and another liquid handling device capable of dispensing into 1536-well densities.
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| Figure 5. Heat map comparison of EL406 and other liquid handler dispensing ECMs into 1536-well biosensor microplate and precision attributable to 384 individual wells for the ECMs fibronectin, collagen, and laminin used in this study. |
CHO-K1/HEK-293 Cell Addition
The final step in label-free cell-based assay preparation is addition of the cells expressing the drug target of interest. For GPCR assays, the most common cell lines used for screening purposes are CHO-K1 and HEK 293 slimming pills cells. Figure 6 demonstrates PWV shift for both CHO and HEK 293 cells added to the ECM-coated wells of the 1536-well biosensor microplate.
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| Figure 6. PWV shift from CHO and HEK 293 addition on uncoated and various ECM coated wells of a 1536-well biosensor microplate. |
As with the ECM-coating, the addition of cells induced large PWV shifts: 300 pm < PWV shift < 1700 pm indicative of good adherence of the cells to the biosensor microplate. Large teeth whitener reviews significant differences in PWV shift are seen between cell type and ECM coating.
Comparisons were also made between the intra-plate reproducibility of PWV shift using EL406 and the same liquid handler used in the ECM deposition. Table 1 depicts the comparisons over 384-wells for CHO deposition for fibronectin.
A significant improvement in precision is evident for CHO cell capsiplex addition across the plate for the ECM’s fibronectin and collagen.
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| Table 1. Precision comparison of EL406 and other liquid handler dispensing CHO cells into 1536-well biosensor microplate with ECM coatings fibronectin and collagen. 384 individual wells were use to determine %CV. |
Assay Validation
The validation of a CHO cell label-free assay prepared by ECM and CHO cell dispensing with the EL406 was performed with ATP-dosing. Extracellular ATP acts as a general cell slimming reviews membrane receptor agonist leading to the stimulation of various pathways and ultimately morphological changes in adhered CHO cells that can be detected by label-free technologies, such as the BIND SCREENER as PWV shifts. Figure 7 shows the PWV shifts associated with ATP stimulation kinetically.
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| Figure 7. Temporal response of CHO cell stimulation with saturating amounts of ATP dosing. |
It is apparent that the ECM fibronectin provides the maximal PWV shift response to ATP stimulation. Other applications will require separate selection of cell line type and ECM for optimal performance.
Conclusions
The EL406 serves as a useful tool for the preparation of label-free, cell-based assays in a 1536-well density. Satisfactory precision is evident for both extracellular matrix deposition and adherent cells allowing for reproducible GPCR assays. The use of 1536-well densities serves as a useful, cost-reduction measure allowing label-free assays to be used economically in primary screening applications.
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