BioFlux Custom Substrate Plates

Instruction for use. Use with BioFlux custom material plates (Catalog No. 910-0119, 910-0120)

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Intended Use

These BioFlux plates are intended for use with any customer-provided material coupon to study the influence of substrate materials on bacterial attachment, bacterial/fungal biofilms, or mammalian cell growth. They are based on 48-well plates and compatible with BioFlux 200, BioFlux One, BioFlux DCIS, and 1000Z systems.

This protocol is used to prepare and prime BioFlux custom substrate plates and remove air bubbles in the channels.

Each flow channel in the Bioflux custom substrate bottom plate consists of two inlet and two outlet wells. Thus a 48-well plate has a total of 12 zones for attaching the custom substrate bottom material. Adhesive film surrounds the substrate access port, allowing for easy attachment of the custom substrate.

The flow path may trap small bubbles during priming. It is critical to remove these air bubbles by following the procedure in Step 2, Priming, of these instructions.

Components

  • BioFlux 200 48-well Custom Bottom plate, 910-0114 (12 experiments / plate)
  • BioFlux 1000 48-well Custom Bottom plate, 910-0120 (12 experiments / plate)

WARNINGS AND PRECAUTIONS

The Custom Bottom plates have been validated for use with coverslip glass coupons that are clean and dry. Use with any material must be validated by the user. Always prime the plates on the lab bench to ensure that the coupons are firmly adhered and do not leak.

Step 1- Attachment of the custom substrate

  • Remove the custom material plate from its packaging
  • Note: each experiment takes 2 wells (2 input, and 2 output to execute). Please refer to the schematic below:
  • Also note that at this point the main channel has an open bottom as shown below:
  • There is one adhesive square around each access port that can be removed (four adhesive backing strips are shown removed below, which will be used for 4 experiments). Note that channels not in use must be completely sealed with plate sealer film.

    Remove the adhesive release paper from the desired zones (e.g., channels 1, 2, or channels 3, 4, etc.). Work the corners of the release paper to start removal. Use a knife blade or tweezers if necessary. If any part of the adhesive starts to pull up, press down on that section, using the release paper to protect the adhesive.
  • At this point select the substrates (in this case we used 170um glass cover slip) and place them onto the desired number of experimental regions, using either tweezers or a gloved hand. The coupon size should be about 25x25mm (5mm accuracy). The surface should be dry and flat to ensure good adhesion.

    The custom substrate plates have been validated with clean, dry 170 um glass cover slips. Use of other materials must be verified by the user. Always confirm that the coupon has firmly sealed without leaks before placing the plate on a microscope.
  • Each coupon should be centered on the round access port as shown below, covering the exposed adhesive layer:
  • After placement of the substrate coupon, apply firm hand pressure to each coupon for at least 10 seconds. Be cautious not to break the glass cover slip. With transparent substrates, you may notice that a pressure sensitive bond is created around the punch hole.
  • The adhered substrate will have now closed the access port as shown below:
  • The adhesive will form a very strong bond that will be hard to de-bond without tearing off the PDMS bottom layer
  • Your device is now ready for priming.

Step 2 - Channel Priming

  • Load 100 uL of PBS or media in each of the two inlet wells and the lower outlet well as shown below (shaded green).
  • Perfuse from inlet to outlet at 20dyn/cm2 for 40 seconds. This priming step should be performed on the lab bench to prevent damage or contamination from leaking coupons. Visually inspect the bottom of the plate after the priming to verify that all coupons are firmly sealed.
  • Inspect visually to see if the channels are filled with fluid and void of air bubbles. A microscope may be used to aid in observation- but only after it has been confirmed that the coupons are firmly sealed and there is no leaking.

    Perform the next steps if significant bubbles remain.

Removal of Air Bubbles

  • Small air bubbles can be eliminated by pressurizing both the inlet and outlet wells at 20dyn/cm2 for 30 minutes or until all the bubbles disappear. This is done by splitting the inlet well air pressure of the line from the BioFlux controller prior to connecting to the interface. The split ends then should be connected to the inlet and outlet of the same column. In the illustration below, column t has been split to go to both columns 1 and 2.

    To split the tubing, using a standard ¼" or 6mm push-to-connect tee fitting and
    appropriate lengths of ¼" or 6mm tubing.
  • Pressurize the channels for 30 minutes or until all air bubbles are eliminated. The plate is now primed and ready for use.

General Tips and Important Notes

  • These plates are not intended for precisely controlled shear experiments, but rather to study surface effects in a flowing environment. For precise shear control, use the standard glass bottom 6-well, 24-well, and 48-well plates.
  • The volumetric flow over the custom substrate is double the indicated flow rate, since flow is occurring from two inlet wells at the same time. However, the actual shear stress is approximately 20% of the indicated shear stress, due to the much larger volume in the region where the custom substrate is exposed to flow. Make sure to account for these adjustments when setting up your experiments.
  • Coupon removal post experiment: To remove coupons, carefully cut around the coupon with a sharp blade, cutting through the PMS to the polystyrene plate. Then carefully pry the coupon up, wearing eye protection and exercising caution against breaking coupons. Additional cutting in the horizontal direction may be needed.
  • Good luck with your material-specific BioFlux experiments! You should also read through our related application note: "Consumables for custom material - biofilm interactions studies under flow". If you encounter any issues, please have a look at the videos on our support website (support.fluxionbio.com) or contact Fluxion support.