The Dianthus 384-well plate with a working volume of 18-25 µL. It is made of black polystyrene material, ensuring that samples are not subjected to sunlight and do not bleach 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 advantage that the plate's top 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. Please also see the article on Liquid Handling to learn more about ideal liquid handling procedures in Dianthus 384-well plates.

Plate sealing:

It is strongly recommended to seal the Dianthus microwell plates (cat. no. DI-P001) to avoid evaporation during incubation times and measurement, and to prevent dust particles to enter the wells. 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))

Of course, there are also automated heat sealers on the market that can be used. We recommend carefully establishing the sealing protocol when sealing Dianthus plates for the first time.

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, we recommend pierceable sealing foil:

• 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, however, not affect the measured 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 Support page.

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

• Keep the plates in the box, closed with tape and away from dust.
• Do not leave fingerprints on the foil bottom. In case, wiping the bottom with a lint-free tissue can remove fingerprints.
• Avoid dust and scratches on the plate bottom. Scratches and dust influence the measurement. An air gun or an air blower blaster pump is recommended to clean the bottom of the plates. Alternatively, wiping the bottom with a lint-free tissue can remove dust.
• 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 when possible (e.g., when dispensing the buffer for a dilution series).
• In general, avoid air bubbles at the plate bottom. This can affect the measurement. It is recommended to centrifuge the plate open (i.e. without seal) at a speed of 460 x g for 2 minutes to eliminate bubbles.
• For a few samples, pipette in tubes and then transfer to the plate using reverse pipetting. In tubes, it is easier to see while pipetting. Therefore, results are often more accurate.
• When pipetting directly and manually in the plate, avoid piercing the foil bottom with your pipette tip.
• One should always work within a volume range of 18-25 µL. 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 fridge or in an incubator with significantly different temperature than the device temperature it is advisable to incubate the plates 30 min before measuring at device temperature.
• Do not freeze the plates.