FAQ - Microtiter plate format

Which side of the MPX slide/plate is the coated?

The MPX slides/plates with the following surfaces: Epoxy (E), Aminosilane (AStar & A+), or Aldehyde (AL) are coated on both sides of the glass. The correct side of the slide/plate for spotting is the same side as the black patterning. The slide/plates that are coated with the 3-D thin film polymer are only coated on one side (the side with the black patterning).

What is the recommended minimum distance from the array to the edge of the patterning?

We recommend that arrays are printed at least 550 µm away from the edge of the patterning. Using this distance with a conventional contact printer and 50 µm diameter pins at a pitch of 150 µm, it is possible to print an array of 40 x 40 spots within each well.

What difficulties are caused by printing the arrays too close to the black PTFE well patterning?

The well edges in the PTFE patterning have a maximum deviation of ± 100 µm. The printer software should be set up to print the arrays at least 550 µm away from the “theoretical” well edge. If the pins are set to print any closer there is a risk of printing over the patterning. Printing too close to the well edges may also cause problems during the hybridization / incubation step. For example, if the silicone superstructure is not accurately placed, it is possible that some spots may be very close to, or even under the superstructure. In this case it will be impossible to hybridizing or scan these spots.

How can I avoid cross contamination between neighboring wells on the MPX slide/plate?

The black patterning is hydrophobic and creates 16/96 square, 7 by 7 mm wells. The combination of the hydrophobic pattering and the self-adhesive silicone superstructure prevents cross contamination between the wells. It is necessary to put the superstructure accurately on the plate patterning, and press down firmly around each individual well to create a good seal. Inaccurately applying the superstructure could result in cross contamination, and also risks introducing adhesive into the printed area:
Packaging of Nexterion Glass Substrates, Plastic Boxes
Cross contamination between wells as a result of an inaccurately applied superstructure

What is the recommended volume that can be added to each well in the superstructure?

The recommended volume per well is between 30 and 130 µl.

For the post hyb washing, is it advisable to remove the superstructure?

We recommend that the silicone superstructure is removed from the plate, as this makes the washing more effective. However, it is possible to leave the superstructure in place during washing, but the results may be suboptimal.

Can SCHOTT provide alternative patterning formats to the 16/48-well MPX slides and 96-well MPX plates?

Yes, this is possible, but will incur additional costs as this will be a customized product. Please provide precise drawings that show the required design.

How can I firmly fix the glass plate into the plate holder during printing and scanning?

The plate can be held in place by four fixing pins in each corner of the plate holder.

How do I locate the A1 well on the MPX-96 glass plate?

There is an orientation chamfer in the black patterning at the corner to indicate the A1 well position.
Chamfer corner in the patterning indication well A1

Which is the recommended spotting solution for the MPX-96 plates?

If the print run is likely to take several hours, then low evaporation print buffers are recommended.
However the best spotting solution to use depends on the coating of the plate:
  • For Plate A+ and AStar NEXTERION® Spot A HD, or 50% DMSO is recommended.
  • Plate E works well with 3x SSC with 0.75 – 1.5 M betaine or NEXTERION® Spot LE.
  • Plate P NEXTERION® Spot PB, or DMSO containing buffers for peptide arrays are recommended.
More detailed recommendations on suitable spotting, blocking and hybridization buffers are available in the individual product protocols.

What is the best way to accurately attach the superstructure to the plate?

Method 1: Using the plate holder to line up the superstructure

Place the printed glass plate into the black plastic plate holder and insert the four corner fixing pins.
Carefully remove the opaque protective backing to expose the adhesive, and apply the superstructure to the plate by holding it at the 4 corners. Once the superstructure is aligned on the plate, press down on each individual well for 3 to 5 seconds to ensure the proper adhesion to the patterned glass.
Method 2: Without using plate holder

Lay the superstructure upside down on clean flat surface (i.e. so the adhesive layer is uppermost). Then remove the opaque protective backing from the superstructure to expose the adhesive. Turn the printed plate upside down, and carefully place it onto the superstructure.
Important: Make sure that the superstructure is not misaligned with the wells on the glass plate, otherwise it will not fit back into the plate holder. Please press the superstructure around each individual well for 3 to 5 seconds to ensure proper adhesion. Fix the glass plate and attached superstructure into the plate holder.
Tecan Hydroflex™

SCHOTT’s R&D team has successfully used the Tecan Hydroflex™ with the NEXTERION® MPX-96 plate.

The best method to remove the superstructure from the plate is to grab the superstructure with tweezers at one corner and gently pull it up and off. During removal take care that all the adhesive is removed from the plate. Also see the question below.

Is there an optimal temperature for removing the superstructure from the MPX-96 plate?

The optimal temperature for successfully removing the superstructure from the patterned glass plate is between 31 and 41°C.
At lower temperatures (20 to 30°C), the double-sided adhesive foil attached to the silicone superstructure can delaminate, resulting in two components.
At temperatures above 41°C the adhesive becomes very tacky, and there is a risk of filaments being created during the removal of the superstructure. These filaments might contaminate the arrays and adversely affect the subsequent image analysis.

How can I avoid evaporation of the hybridization solution during the hybridization?

To avoid evaporation of the hybridization solution, it is important that the clear sealing sheet supplied with the 96-well superstructure is used to seal the top of the wells during processing. If the hybridization is likely to exceed one hour, we also recommend the use of a dedicated hyb chamber. A suitable product is available on request from SCHOTT.
Hyb chamber from Schott (available on request)

Can the MPX-96 kit be used more than once?

The MPX-96 coated plate, and superstructures are designed for single use only, and should not be re-used. However, the black plastic plate holder and pins may be cleaned and reused with a new coated plate and superstructure. If not all 96-wells are required for an assay then we recommend using the 16-well NEXTERION® MPX-16 slides instead. These multi-well slides are available with the same coatings as the MPX-96 plates.
Dimensions of NEXTERION® MPX slides
Dimensions of NEXTERION® MPX 4 tray
One issue to be aware of, is that the four slide holders in the MPX-4 tray are slightly larger (at 75.9 mm x 25.3 mm) than the MPX-16 slides themselves (which are 75.6 mm x 25.0 mm.

Over what temperature range can I use the MPX 96-well silicone superstructures?

The 96-well silicone superstructures have been successfully tested at temperatures between 20 and 65°C using typical hybridization buffers.

What types of reagents are the MPX 96-well silicone superstructures compatible with?

The 96-well silicone superstructures have been successfully tested with typical aqueous based hybridization buffers, including one containing 50% formamide (methanamide).

What is the recommended minimum and maximum buffer volume that I can add to the wells of the NEXTERION® MPX-96 microplate?

The dimensions of the superstructure wells are 7.0 by 7.0 mm with a depth of 4.5 mm.
The minimum volume that will cover the bottom of the well is: 40 microlitres (PBS buffer with detergent) or 50 microlitres (PBS buffer without detergent).
The maximum recommended „safe“ working volume is: 130 microlitres (PBS with detergent) or 160 microlitres (PBS without detergent).
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