96 well microtiter plate

The titers of M2e-specific antibodies were detected by ELISA as described previously.^{24 (link)} Briefly, a 96-well micro-titer plate (Sigma, St. Louis, SO, USA) was coated with either H5N1–, H7N9–, or HK/156-M2e (GL Biochem Ltd, Shanghai, China) at a concentration of 1 µg/well and incubated overnight at 4 °C. After the coated plate was blocked with 3% bovine serum albumin in PBS for 2 h at room temperature (RT), serially diluted sera were added to the plate and incubated at RT for 2 h. Horseradish peroxide-conjugated goat anti-mouse immunoglobulin G antibody (Dako, Glostrup, Denmark) was added to the plate and incubated at RT for 1 h. Substrate 3,3′,5,5′-tetramethylbenzidine (Life Technologies, Carlsbad, CA, USA) was added to the plate and incubated at RT for 0.5 h. The reaction was stopped by adding 1 M H₂SO₄, and the results were measured at absorbance of 450 nm using an ELISA reader (Beckman Coulter, Brea, CA, USA).

Gp120 proteins produced from 293F cells were coated onto 96-well microtiter plates (Sigma-Aldrich, St. Louis, MO, USA) at 1 µg/mL in 100 µL of phosphate-buffered saline (PBS) as previously described.^{30 (link)} Plates were washed five times with PBS containing 0.1% Triton-X (EWB) and blocked overnight at 4 °C in PBS containing 4% whey and 5% powdered milk. R53, biotinylated anti-rabbit secondary antibody (Vector Labs BA-1000) at 1.5 µg/mL, and a streptavidin horseradish peroxidase construct (Vector Labs SA-5004, Burlingame, CA, USA) at 500 ng/mL were added sequentially to the wells in a volume of 100 µL. Plates were incubated for 1 h at room temperature and washed five times after each step with EWB and then developed for 3 min in 100 µL of a 3,3′5,5′-tetramethylbenzidine substrate solution (Sigma-Aldrich T3405, St. Louis, MO, USA). The reactions were stopped with 25 µL of 2N H₂SO₄.

The MIC of tea tree and rosemary oils were determined using a broth assay (Klančnik et al., 2010 (link)) in 96-well microtiter plates (Sigma Aldrich, USA). Fresh cultures of the tested isolates were prepared in Brain Heart Infusion (BHI) broth, inocula of concentrations 2 × 10⁷ cfu/ml were used. A two-fold dilution series of tea tree and rosemary oils were prepared in 1% DMSO to yield final concentrations ranging from 5000 mg/L to 9.7 mg/L. Chloramphenicol was employed as a positive control. After 18 h and 48 h (for C. acne), the optical density at 600 nm was measured with a microplate reader (680 XR reader, Bio-Rad). Bacterial growth was confirmed by adding 10 μl of a sterile 0.5% aqueous solution of triphenyltetrazolium chloride (TTC, Sigma–Aldrich) and incubating at 36 °C for 30 min. The viable bacterial cells reduced the yellow TTC to pink/red 1,3,5-triphenylformazan (TPF). All assays were performed in triplicate. Streaks were taken from the two lowest concentrations of each oil concentration exhibiting invisible growth and were sub-cultured onto Blood agar media. The plates were incubated at 37 °C for 24–48 h, then inspected for bacterial growth in corresponding to both the oils. MBC was taken as the concentration of the oil that did not exhibit any bacterial growth.

E3 medium (embryo cultivation medium) was freshly prepared prior to use. It consisted of deionized water, 5 mM NaCl, 0.17 mM KCl, 0.33 mM CaCl₂ and 0.33 mM MgSO₄. The salts for the medium preparation and other chemicals such as 96% ethanol p.a., isopropanol p.a. and trypan blue were provided by Sigma-Aldrich s.r.o., Czech Republic. Other material used for the experiments was: 1.6 mm ID silicone tubing (ibidi GmbH, Germany), 1/16′′ OD × 1/32′′ ID PTFE tubing (Darwin Microfluidics, France), 1/8′′ OD × 0.062′′ ID PFA high purity tubing, 1/4–28 fittings with ferules and manual shut-off valves, debubblers and solvent caps for 1/4–28 fittings, all provided by Cole-Parmer Instrument Company Ltd, Great Britain. For the FET cultivation experiments, 96-well microtiter plates (Sigma-Aldrich s.r.o., Czech Republic.) and a home-made thermobox with Arduino-based PID temperature control (incubator) were used.