Clear polystyrene 96 well microplates

Ninety-six-well plates (Corning Clear Polystyrene 96-Well Microplates) were coated overnight with HDM extracts at 200 μg/ml in 0.05 M Na2CO3 pH 9.6. The coated plates were then blocked for 1 h with 1% BSA in PBS. Serum from mice was collected and serially diluted (threefold) in PBS with 10 mg/ml BSA and 0.1% Tween 20 before incubation in the coated plates. After washing, bound antibody was detected with HRP-conjugated goat anti-mouse IgE (1:2,000, 1110-05; Southern Biotech, Birmingham, AL, U.S.) and quantified by spectrophotometry at 405 nm (OD).

Chlorhexidine digluconate (CHX) was from Sigma-Aldrich. BD™ Bacto™ brain heart infusion, BD BBL dehydrated brain heart infusion agar, and Corning™ Clear polystyrene 96-well microplates, Corning™ 3370 were obtained from Fisher. AlamarBlue assay kit was obtained from Fisher.
Triethylene glycol dimethacrylate (TEGDMA), 2-hydroxyethylmethacrylate (HEMA) and 2-methacryloyloxyethyl phosphorylcholine (MPC) were used as received (Sigma-Aldrich) without further purification. Camphorquinone (CQ), ethyl-4-(dimethylamino) benzoate (EDMAB) and diphenyliodonium hexafluorophosphate (DPIHP) were obtained from Sigma-Aldrich (St. Louis, MO, USA) and used as a three-component-photoinitiator system without further purification. ε-pL hydrochloride was purchased from Bonding Chemical (Katy, TX, USA). To obtain ε-pL), 10 g ε-pL hydrochloride was dissolved in 60 mL sodium bicarbonate buffer (NaHCO3, 1.5M) and dialyzed with membrane (Spectra/Por®7 Dialysis Tubing, MWCO 1kD) in deionized water for 4 days. Then the solution was dried at 37 °C and ε-pL was obtained.

Strains containing different EGFP expression cassettes were analyzed with a fluorescence microscope (Olympus IX73; Olympus, Tokyo, Japan). First, an image with a magnification of 40 × in bright filed (BF) was photographed. Subsequently, EGFP fluorescence was observed and photographed using the channel of fluorescein isothiocyanate (FITC) with the excitation (ex) at 490 nm and the emission (em) at 518 nm. An automatic exposure time was set. In parallel, a 200 μL culture of R. toruloides containing different EGFP expression cassettes were transferred to Corning^™ Clear Polystyrene 96-Well Microplates and Costar^® 96-Well Black Polystyrene Plate (Corning Incorporated, USA) for the measurement of biomass and fluorescence. The biomass and fluorescence were measured using EnSpire^® Multimode Plate Reader (PerkinElmer, USA) under 600 nm or 485(ex)/525(em) nm, respectively. The fluorescence signal was normalized to the cell density to make the data comparable.