Plates

The Dianthus 384-well plate has a working volume of 18-25 µL. It is made of black polystyrene material which ensures that samples are not subjected to sunlight and do not photobleach easily. The bottom of the Dianthus plate is made of transparent material as the Dianthus optical system measures the sample from the bottom. This has the added advantage that the top of the plate can be sealed to avoid sample oxidation and evaporation. The plates are barcoded with a unique barcode that is recognized by the Dianthus instrument. The plates are coated with a special polymer to avoid protein adsorption to Well walls and well bottom. Aso see the article on Liquid Handling to learn more about ideal liquid handling procedures in Dianthus 384-well plates.

Plate sealing

For TRIC measurements in Dianthus™ NT.23 instruments it is strongly recommended to seal the Dianthus microwell plates (cat. no. DI- P001) before loading them into the instrument to avoid evaporation during the measurement and incubation times. There are multiple plate sealing options available on the market. Here are just a few examples:

Heat sealing

Heat sealer:

• Thermo Fisher Scientific®, ALPS 50 V-Manual Heat Sealer™ (cat. No. AB-1443A)

Heat- sealing foils (optimal sealing parameters for both heat- sealing foils: 165°C, 2.5 seconds):

• Sigma®, PierceASeal Foil™ heat sealable seal, (cat. No. Z740334-100EA (non- peelable))
• 4titude®, Polystyrene Foil™ Heat Seal, (cat. No. 4ti-0547 (peelable))

There are also automated heat sealers on the market that can be used. Carefully establishing the sealing protocol when sealing Dianthus plates for the first time is recommended.

Glue sealing

• 4titude®, PCR Foil Seal, 4ti-0550
• Greiner Bio-One, SILVERsealTM, 676090

During lengthy liquid handling steps, where evaporation should be prevented but the wells should still be accessible to pipetting solutions, pierceable sealing foil is recommended:

• 4titude®, Pierceable Seal, 4ti-05666/384

Re-using wells

Wells can be measured multiple times but should not be used multiple times with different samples.

Protein adsorption

The wells are coated with a polymer to avoid protein Adsorption to the plate surface. If adsorption to the plate material does occur it will result in a decreasing initial fluorescence value over time, which should not affect the measured TRIC response. A decreasing initial fluorescence value over time can easily be tested with the time-course experiment templates.

Well geometry

The wells are conically shaped, round and have a flat bottom. A document with exact measures can be obtained from the NanoTemper Explorer Community.

Barcode

The barcodes printed on a sticker on the plates are unique and encrypted and can only be de-crypted in Dianthus instruments.

General tips for handling plates

• Do not leave fingerprints on the bottom of the plate. If this occurs wipe the bottom with a lint- free tissue to remove the fingerprints.
• Avoid dust and scratches on the bottom of the plate. Scratches influence the measurement, while dust generally does not. Dust should still be avoided, clean the bottom of the plate with a lint-free tissue if needed.
• Use reverse pipetting where possible to avoid air bubbles. If you pipette manually in a 384-well plate it is advisable to use reverse pipetting in cases where it is possible (e.g. when dispensing the buffer for a dilution series).
• In general, avoid air bubbles at the bottom of the well. This can affect the measurement. If air bubbles appear, centrifuge the uncovered plate (i.e. without seal).
• If working with few samples, prepare them in tubes and then transfer to the plate using reverse pipetting. In tubes it is easier to visualize bubbles, therefore results are often more accurate.
• When pipetting directly and manually in the plate, avoid piercing the bottom of the well with the pipette tip.
• A volume of 18-25 µL should be used. In this range small volume errors will not affect the measurement. 25 µL is the maximum volume a well can hold. The optimal recommended working volume yielding high quality data is 20 µL.
• If plates are stored in a refrigerator or an incubator with significantly different temperature than the device temperature it is advisable to incubate the plates 30 min before measuring at the device temperature.