12 well plate

Cell lines cultivated for the allergen treatment included HEK293 and HeLa. HEK293 cells were cultivated in Dulbecco’s modified Eagle medium (DMEM, Sigma-Aldrich, St. Louis, MO, USA) supplemented with 10% heat-inactivated fetal bovine serum (FBS), 1% pen/strep (penicillin 10,000 U/mL; streptomycin 10 mg/mL) and 200 mM L-glutamine (Sigma-Aldrich, St. Louis, MO, USA), and HeLa cells were cultivated in Eagle’s minimum essential medium (EMEM, Lonza, Basel, Switzerland) supplemented with 20% FBS, 1% pen/strep (penicillin 10,000 U/mL; streptomycin 10 mg/mL) and 1% (v/v) 200 mM L-glutamine. Cell lines were grown in a humidified atmosphere of 95% air and 6% CO₂ at 37 °C, in T-25 flasks (Thermo Scientific, Waltham, MA, USA) until confluence and then were trypsinized (0.25% Trypsin-0.53 mM EDTA). HEK293 (250,000 cells per mL) and HeLa (300,000 cells per mL) cells were seeded at a volume of 1 mL in 12-well plates (Sarstedt, Nümbrecht, Germany) and grown to confluence before allergen treatment.

The OA potential toxicity (715 µg/mL) was examined in the zebrafish embryo acute toxicity test in accordance with the Organization for Economic Cooperation and Development (OECD) recommendation for the testing of chemicals (Test No. 236 [107 (link),108 ]). Zebrafish embryos were screened for viability, shape, and transparency one hour after fertilization. Only the eggs meeting all criteria, i.e., being viable, completely transparent, and round, were chosen and moved to 12 well plates (Sarstedt, Germany). They were randomly divided into two groups, the control and experimental ones, and kept in each well until the time of experiment elapsed, i.e., up to 96 h. Two batches of embryos (3 wells per batch, n = 5–7 per well) were kept in 3000 µL of the zebrafish medium (control group) or supplemented with 715 µg/mL of OA (experimental group). The mortality rate was scored after 23, 47, 71, and 95 h of incubation. Hatchability was assessed after 71 and 95 h of incubation. The morphological abnormalities were assessed after 95 h of incubation: heartbeat, heart/yolk edema, yolk sac necrosis, hemorrhage [108 ], jaw development, eye size, and posture. Additionally, for assessing muscle function and performance, the touch-evoked response assay was conducted as described in detail previously [109 (link),110 (link)].

BOECs were seeded on 18 mm coverslips in 12-well plates (Sarstedt, Numbrecht, Germany). Cells were fixed with 4% paraformaldehyde (PFA) (Electron Microscopy Sciences, Hatfield, PA, USA) in phosphate-buffered saline (PBS) (Wisent Bioproducts, St. Bruno, QC, Canada). Immunofluorescence staining and imaging were done according to the manufacturer’s protocol (Thermo Fisher Scientific, Waltham, MA, USA). Rabbit anti-VWF (DakoCytomation, Glostrup, Denmark) and goat anti-VECAD (Santa Cruz, Dallas, TX, USA) dilution 1:100 were used as primary antibodies. Corresponding species-specific Alexa Fluor 488- and Fluor 555-labeled antibodies (Thermo Fisher Scientific, Waltham, MA, USA) dilution 1:500 were used as secondary antibodies. Images were recorded by spinning disk confocal microscopy (Olympus IX81, Olympus corporation, Tokyo, Japan) under control of Volocity software (PerkinElmer, Groningen, The Netherlands).

Cells were seeded on 12-well plates (Sarstedt, Wr. Neudorf, Austria) at an initial density of 1 × 10⁶ cells/mL. After 24 h, the cell suspension was stimulated with coumarin-derivatives at concentrations ranging from 10 µM to 1000 µM. After 24 h, 1 mL of cell suspension was centrifuged at 1000 rpm for 5 min and the supernatant was discarded. The cells were resuspended in 50 µL PBS. From each tube, a 10 µL-cell suspension was taken and mixed with 10 µL of Trypan blue reagent (Bio-Rad, Hercules, CA, USA). The sample was incubated for 5 min. Cell viability was measured with a TC20 Automated Cell Counter (Bio-Rad, Hercules, CA, USA). Each experiment was repeated three times.

Tolerance to desiccation was studied by keeping (RT, 6 days) a suspension (200 μL/well, 10⁹CFU/ml, TSB) completely dried by evaporation into 12-well plates (Sarstedt) by triplicate and rehydrating the pellet in PBS to determine the viable CFU/ml in BAB. Susceptibility to detergents was firstly evaluated with SDS (Merck, Darmstadt, Germany), since this surfactant inactivated all the Brucella tested at 0.06%; bacterial suspensions (10⁴CFU/ml, 100 μL, by triplicate) were cultured in BAB with 0.1% Triton X-100 (Sigma-Aldrich, San Luis, MO, United States). Oxidative stress and acid pH resistances were analyzed by incubating (37°C, 1 h) a 10⁴ CFU/ml suspension in TSB with an equal volume (100 μL) of H₂O₂ at increasing concentrations up to 5 mM or with acidified TSB at a final pH from 7.3 to 2.3 by duplicate. Results were expressed as bacteria (%) vs. control.

Cytochrome P450 (CYP) 3A4 activity was measured using the CYP3A4 P450-Glo™ assay (with Luciferin-IPA) (Promega) as previously described [13] (link). Briefly, HeLa cell lines were seeded at a density of 2×10⁴ cells/well in 12-well plates (Sarstedt Inc.). Twenty-four hours later, 0.5 µg of CYPOR-YFP plasmid (kindly provided by Dr. Byron Kemper, University of Illinois, USA) was transfected into cells using Lipofectamine 2000 reagent (Life Technologies). Twenty-four hours after transfection, cells were washed with phosphate-buffered saline and fresh media containing luminogenic substrate (50 µM) was added. After a 3 h incubation period, 100 µl of media from each well was mixed with an equal volume of Luciferin Detection reagent and luminescence was measured using a LUMIstar luminometer (BMG LABTECH).