Aeraseal film

Matrigel matrix (Corning, Inc., Corning, NY, USA) was incubated overnight on ice at 4 °C prior to the assay. On the day of the assay, the Matrigel matrix was transferred into a 96-well plate and allowed to polymerize for 1 h at 37 °C. The HUVEC suspension (150 µL) at 2 × 10⁵ cells/mL was seeded and incubated for 2 h at 37 °C and 5% CO₂ to allow the HUVECs to attach onto the Matrigel. An additional 200 µL of medium was added to each well and the plate was sealed with AeraSeal film (Sigma, St. Louis, MO, USA). Group 1 G and taSMG were grown for 18 h in a humidified 37 °C, 5% CO₂ incubator. Group 1G-to-taSMG was incubated under 1 G for 9 h and then incubated under taSMG for another 9 h. Arrangement of the endothelial cell was observed with an Eclipse Ts2 (Nikon, Tokyo, Japan) microscope under 4x magnification.

For fed-batch cultivations, 96-square-well (FeedPlate®, Part number: SMFP08002, Kuhner Shaker GmbH, Herzogenrath, Germany) and 48-round-well (prototype) microtiter plates were used. At the bottom of each well, a release system is located, which consists of a cross-linked siloxane elastomer with embedded glucose crystals. The cross-linking is catalyzed by a Karstedt’s catalyst. The embedded glucose serves as a reservoir and is released by an osmotic driven mechanism. For the prototype, the permeation capability of the siloxane matrix for water is enlarged by the addition of hydrophilic siloxane copolymers. For batch cultivation, 96-square-well (Part number: 850301, HJ-BIOANALYTIK GmbH, Erkelenz, Germany) and 48-round-well microtiter plates (MTP-R48-OFF, m2p-labs GmbH, Beasweiler, Germany) were used.
For all microtiter plate cultivations and glucose release experiments in 96-square-well plates, “AeraSeal Film” (A9224, Sigma-Aldrich Chemie GmbH, Germany) sealings were used as permeable sterile barrier [40 (link)]. For release experiments at 45 °C, an airtight, self-made silicone sealing was fixed on top of the plate. The whole setup (microtiter plate and sealing) was placed inside the cultivation hood. “Polyolefin sealing foil” (900371-T, HJ-Bioanalytik GmbH, Erkelenz, Germany) was used for cultivations in 48-round-well plates.

All bacterial isolate cultures were diluted (1:20) in their respective media supplemented with 0.5% D-glucose (w/v). The bacterial solution was pipetted across two or three columns of a 96-well microtitre plate at 150 µL per well alongside control wells (TSB/THB–D-glucose). The plates were covered with AeraSeal film (Sigma, St Louis, MO, USA) and incubated with shaking (50 rpm) at 37 °C in air for 48 h, refreshing the wells with fresh media and D-glucose after the first 24 h.
The media and any non-adherent planktonic bacterial cells were carefully removed by a pipette and the wells were air-dried for 20 min. The adherent cells and biofilm (i.e., biomass) were stained using 150 µL of 0.2% (w/v) crystal violet (CV) and 1.9% ethanol and incubated at room temperature for 10 min. The solution was then removed and the wells were carefully washed twice with PBS to remove the unbound dye. The bound CV was solubilised using 150 µL of 1% SDS for 10 min at room temperature and then vigorously agitated. The solution was plated at a 1:5 dilution in 1% SDS and the absorbance read at 540 nm (A540). The assays were performed in triplicate for each bacterial isolate. The strength of the biofilm formation was categorised as: A540 < 0.5, non-biofilm-forming; 0.5–1.5, weak; 1.5–2.5, intermediate; 2.5–3.5, strong; and A540 > 3.5, very strong [58 (link),59 (link),60 (link)].

90 μL of a culture prepared as above and resuspended in M9 media supplemented with 730 μM ceftazidime to an OD_{600 nm} of 0.8 was treated with two-fold dilutions of compounds in DMSO (in a final DMSO concentration of 0.016% (v/v)). Samples were incubated in a 96 well black walled plate covered with AeraSeal film (SigmaAldrich) at 28°C for 24 hours before quantification of viable cells with PrestoBlue as above. IC₅₀ values were fitted to Eq (2) using Graphpad Prism version 6.0.1.

Mites were identified as fed when they were engorged and bright red. They were considered dead if they did not show any movement and were unresponsive to touch stimulus. Engorged mites were collected from the pouches and transferred into individual wells of a 96-well tissue culture plate (Costar, Corning, NY, USA) and sealed with AeraSeal film (Sigma-Aldrich, St. Louis, USA; A9224-50EA). Plates were placed into an incubator (25 °C and 85% relative humidity) and checked every 24 h for five days and on day 7 after feeding. Feeding rate was recorded when transferring from the pouch to the plates. Mite mortality, laying mites, oviposition, egg hatchability and larval development were recorded in every check to assess the vaccine efficacy.