96 well plate

The xylanase potential of TpABF43_36b was evaluated using insoluble AZCL xylan. AZCL-xylan (beechwood) was suspended in buffer 50 mM NH₄Ac, 50 mM KCl, 0.01% Triton X-100 pH 5.0 (pH adjusted with 2M HCl) while stirring (0.2% w/v). Enzyme incubations were performed with 90% (v/v) substrate suspension and 10% (v/v) TpABF43_36b (diluted 1, 5, and 10×) in single determination in 96 well plates (Thermo Fisher Scientific, Waltham, Massachusetts, USA) while shaking (600 rpm) for 1 h at 40 °C with a total volume of 250 μL. AaXyn10 was used for reference with 1 to 1024× dilutions. After incubation, the plate was centrifuged at 2000× g rpm for 2 min. Subsequently, 100 μL supernatant was added to a 96 well plate (Thermo Fisher Scientific, Waltham, MA, USA) and measured at 595 OD (any solubilized AZCL-xylan fragments give absorbance at this wavelength) using a SpectraMax Plus 384 microplate reader (GE Healthcare, Chicago, IL, USA).

In order to assess proliferation and apoptosis (during the proliferation stage) progenitor cells were plated on 96-well plates (Nunclon) at a density of 1.2 × 10⁴ cells per well per well in 100 μl RMM media. Cells were cultured in the presence of EGF, bFGF and 4-OHT for 24 h followed by 2 days incubation with 1% serum samples (the same concentration previously used in the lab as able to induce a profound effect without causing over-confluence or any alteration in the composition of neurons) from depressed or non-depressed IFN-α-treated HCV patients, also in the presence of growth factors containing 0.5 mg/ml penicillin streptomycin. The synthetic nucleotide bromodeoxyuridine (BrdU) (B5002, Sigma) with a final concentration of 10 μM was added to the media 4hrs before the end of the incubation to label proliferating cells.
Whereas, in order to assess neuronal differentiation and apoptosis (during the differentiation stage) cells were plated on 96-well plates (Nunclon) at a density of 1.2 × 10⁴ cells per well per well in 100 μl RMM media. Following the proliferation phase previously described, cells were washed twice for 15 min in RMM media (without EGF, bFGF and 4-OHT), and then cultured with 1% serum samples from depressed or non-depressed IFN-α–treated HCV patients in RMM containing 0.5 mg/ml penicillin streptomycin for subsequent 7 days.

We first tested the antimicrobial activity of 10.5–330 μg/ml histone type III (histone mixture) by dilution of histone in final volume of 50 μl. The reaction mixtures were added to wells of 96-well plates (Nunc, Denmark) containing 150 μl of B. subtilis PY79 cells (at mid-logarithmic growth diluted 1:5,000 in LB) to complete a 200 μl final volume. For measuring bacterial growth inhibition activity of histone mixture, the plates were transferred to a GENIOS Microplate Reader (TECAN, Austria), and absorbance at 595nm were measured during incubation at 37°C every 20 min (after automated mixing/aeration for 500 s) to generate growth curves. Percent growth inhibition of each treatment was compared with growth at late logarithmic growth phase of untreated bacteria (0% growth inhibition, ∼10 h). To test the effect of actin and DNA on the growth inhibition action of histones, histone mixture (50 μg/ml) were incubated with or without 50–150 μg/ml F-actin or 10–100 μg/ml DNA for 10 min at 37°C, then, 5 μl of the protease-containing late logarithmic growth supernatant of P. gingivalis diluted 1:10 was added (or not) to the 50-μl reaction mixture and incubated for 30 min at 37°C. The reaction mixtures were added to wells of 96-well plates (Nunc, Denmark) containing 150 μl of B. subtilis PY79 cells and growth inhibition was measured as described above.