Rather than using physical wells, DropArray microplates are completely flat including a series of 96 or 384 hydrophilic spots. A hydrophobic surface occupies the entire flat surface area of the plate except for the spots. These spots form an array that conform to the well layout of the ANSI/SBS 4-2004 microplate standard. Liquid dispensed onto these spots form droplets that remain in a fixed position from the surface tension created from the hydrophobic surroundings, even when the plate is shaken or inverted. The DropArray microplate only has walls around the outside of the plate, which serve to contain wash buffers added in bulk during wash steps of the assay. DropArray offers three platforms that address unmet needs for cell processing with flow cytometry (DA-Cell), miniaturizes and improves magnetic bead-based assays (DA-Bead), and enables reduction of cells and expensive reagents for high content screening (DA-HCS).
DropArray microplates enable simple and easy miniaturization of assays at 2-4 microliters for the 384-well format and 5-15 microliters for the 96-well format because of their unique wall-less design. The wall-less feature eliminates capillary action, the biggest barrier to miniaturization with traditional microplates. Furthermore, the wall-less feature allows extremely gentle washing by liquid exchange, which is different from the harsh, convection-based washing performed by a microplate washer with an array of nozzles.
Unlike a standard microplate that requires dispensing and aspiration from each well, bulk liquids like wash buffers are added to or removed from the entire DropArray microplate at once. By reducing turbulence in wash steps, far fewer cells and beads are lost from the assay droplets, even with suspension cells and non-magnetic beads compared to traditional microplates. Evaporation is controlled using an Anti-Evaporation Lid which provides a humidified environment and minimizes evaporation of the droplets.
What pipette do you recommend to handle 5—15 microliters of sample and reagent for DropArray microplates?
Among all the pipettes available in the market, Curiox users find that Rainin® pipette is best at handling small volumes of 5 – 15 microliters, particularly when a solution is sticky or viscous due to the high content of proteins and additives. Rainin pipette tips have a long narrow body and small orifice which minimizes a chance of wicking of a solution onto a tip. Pipettes of other brands and suppliers can be used but they need a bit of skill to precisely handle small volumes.
A DropArray microplate does not require any special skills because the hydrophilic spots on the plate attract reagents while the hydrophobic surface between the spots repels reagents. A dispensed droplet adheres to a spot and does not come off during the typical handling expected for a plate. When dispensing reagents at small volumes for bead-based assay applications or replacing centrifugation for flow cytometry cell processing, DropArray improves workflow over traditional methods.
A DropArray Washing Station typically needs ~110 ml for a 1x washing cycle. A 1x wash cycle and 3x wash cycles need ~2 min and ~4 min respectively, including 30 seconds of built-in rest time needed before starting the next wash process for precipitating beads onto the surface of a plate.
DA-Cell for Flow Cytometry
The minimum time for optimized cell recovery is recommended at 10 minutes. The addition of 10 to 20 minutes of incubation time reduces the loss of cells to less than 0.1% per wash step.
Yes, the DA-Cell plate is compatible with any cytometer that has a 96-well plate loader.
We are interested in rare cells and events and usually need larger volumes. What is the maximum cell number and volume of reagent per well of a DA-Cell plate?
DA-Cell plate can process up to 10 million cells per well without affecting the washing and staining. A DA-Cell plate can hold up to 75 µl of reagent, and this volume can increase to 300 µl by using a simple accessory.
Yes, per your usual protocol the fixing, permeabilization, and staining can be performed all on the DA-Cell plate.
The plate can be run in full or partially. If only a few wells are used, the plate can be stored at 4°C and the remainder of the wells can be used later. However, wells cannot be reused and caution must be taken to document the wells that have been used. Once all the wells are used, they can’t be reused.
How do the cells stay in the well and not get washed away with the laminar flow created by the dual-nozzle action?
DropArray (or DA) plate has a unique patent-protected wall-less design. The absence or minimal presence of a wall allows for laminar flow during the addition and aspiration of a liquid. By nature of a laminar flow, the velocity of the flow reaches zero at the surface of a plate where cells are resting by gravity. Such low velocity allows cells at the surface to remain where they are even if the number of cells is as small as one. Yet, the bulk of existing liquid is replaced by an incoming liquid effectively rinsing the spot and cells.
The entire wash protocol has been optimized to combine the unique, patented features of wall-less plate as well as the flow rate and incubation time to obtain the greatest cell recovery. Changing the flowrate is possible if desired.
The DA-Cell well can hold up to 70 µl and up to approximately 5 million cells. During the protocol, the DA-Cell plate is vortexed and the working volume range above has been validated by our scientist. Volumes above 70 µl are not recommended and may jeopardize the ability of the spot to be retained on the wall-less plate during vortex of a plate.
DA-Bead for Bead-Based Immunoassays
I am using bead-based immunoassays, which use magnetic beads. How would my assay benefit by using DA-Bead microplate?
DA-Bead is a unique washer and wall-less plate system that allows simple and easy miniaturization by simply reducing the volume of samples and reagents by 5 – 10 fold under the same workflow. Alternatively, you may download posters and publications presented by DA-Bead users following the links on this website. For the details please contact a Curiox representative.
It appears that handling 5 – 10 microliters of solutions by manual pipetting is not practical. Wouldn’t it require automated liquid handling instrument to dispense such small volumes, particularly with sticky biological samples?
Handling of 5 – 10 microliters of solutions with a traditional plate or tube is not easy as a traditional microplate is designed to hold a much larger volume. The DropArray DA-Bead microplate is designed to facilitate handling small volumes of solutions specifically. With the help of Rainin pipettes, DropArray users have found that the handling of small volumes is not different from larger volumes for a traditional plate.
Yes. The DropArray HT Washing Station (HT200) has a PC-HT200 RS232C port for communication with any automation controllers.
CVs are a result of variability and this variability can come from the user or the assay. In general, dispensing smaller volumes has more error than dispensing larger volumes, but this is very user/technique-dependent and pipette quality driven. It is known that the higher intra-assay CVs for bead-based assays stem from a combination of steps, mostly from dispensing, washing, and Ab-Ag binding on the bead surface. The DropArray system may produce higher CVs purely due to dispensing in smaller volumes. However, uniform and consistent specific binding with minimal non-specific binding and better washing with minimal loss of beads has been shown in studies to reduce CVs. The overall impact of the lower volume will depend on the specific test conducted and can be analyzed using comparative studies.
Are there enough beads when a bead-based assay is reduced to 5 microliter bead solution on a DA-Bead plate?
Yes. For example, in a 25 microliter bead solution, there are ~2,500 beads per analyte. A 5 microliter bead solution contains ~500 beads per analyte, which is still excessive for the number of beads required for reading, which is generally between 50 and 100.
No. The entire workflow of a bead-based assay on a DA-Bead plate is identical to that of a conventional microtiter plate. The DA plate replaces the traditional microplate and the DropArray Washing Station is used during the washing steps.
DA-HCS for Cellular Assays
No. The DropArray platform allows seamless use on any conventional dispenser, gripper, stacker, reader or imager.
The DropArray DA-HCS plate can be used with a low confluence of 80 cells per mm2 to a very high confluence of 630 cells per mm2. For first time users, we recommend 250 cells per mm2. The cell growth rate on DA-HCS plates is no different compared to a conventional microplate.
Depending on cell-line and assay requirements, cells can survive up to two weeks without media exchange. Media can be aspirated manually or washed off using DropArray HT/LT Washing Stations before applying fresh media.
DropArray DA-HCS plates are tissue culture-compatible. Like a conventional tissue culture plate, DA-HCS plates can be easily coated with DNA, poly-lysine, Collagen, fibronectin, silane-compatible substrates, or any other cell culture coatings. In addition, pre-coated plates for different cellular assays are available.