Ultra 384

Samples for total bacterial abundance (as a proxy for biomass) were taken on a daily basis and preserved by adding formaldehyde to a final concentration of 2%. Cells were stained with SYTO 13 solution (1.25 μM, Molecular Probes)^{61 (link)}, and their abundance was determined using a Cyflow flow cytometer (Partec, Münster, Germany).
Bacterial carbon production was measured every day using leucine incorporation⁶² . L-[4, 5-3H] Leucine (Perkin Elmer) was diluted to 15% with unlabelled l-Leucine (Sigma, St Louis, MO, USA) and added at a final concentration of 100 nM. Samples and blanks were incubated at 13 °C for 1 h. Disintegrations per minute (DPM) was recalculated to bacterial carbon production rates (ng C L⁻¹ h⁻¹)⁶³ .
The activities of cellobiohydrolase and β-glucosidase were measured prior to each disturbance and six days after the pH pulse disturbance. Enzymatic activities were measured using methylumbelliferone (MUF)-linked substrates (Sigma-Aldrich) under saturating conditions (0.6 mM final conc.). The samples, blanks and MUF standards were incubated for 3 h in the dark at room temperature. After incubation, glycine buffer (pH 10.4) was added (1:1 v:v) and fluorescence was measured at λ_ex/em = 360/465 nm (Ultra 384, Tecan, Switzerland)^{64 (link)}.

The cytotoxicity of a test compound was determined, as previously described (Furuta et al., 2002 (link); Han et al., 2011 (link)) by measuring cell viability in the presence of various compound concentrations. Briefly, 100 μl MDCK cells (0.5 × 10⁵ cells/ml) were dispensed into wells of a 96-well plate and incubated at 37°C/5% CO₂ for 24 h. The test compound at the serial dilution in 100 μL of DMEM without serum was added to the cells. After culture for 72 h, cell viability was measured using the Cell Counting Kit-8 (CCK-8), which was purchased from Dojindo Laboratory (Kumamoto, Japan), and using a spectrophotometer (Ultra 384, Tecan, NC, United States). The percent cytotoxicity (=100% – % cell viability) was calculated, and CC₅₀ (half-maximal cytotoxic concentration) of the test compound was determined using the Calcusyn software program (Biosoft, Ferguson, MO, United States) (Chou and Talalay, 1984 (link)).

A sandwich ELISA was conducted to determine the cross-reactivity of the peptides with the preexisting antibodies in HIV-1-infected patients. T20, C46, AP1, AP2 and AP3 were coated onto the wells of 96-well polystyrene plates (Costar, Corning Inc., Corning, NY) at 10 μg/ml. The wells were then blocked with 1% gelatin, followed by addition of 50 μl of serially diluted sera from HIV-1-infected patients and incubation at 37 °C for 1 h. Then, HRP-labeled goat-anti-human IgG (Abcam, UK) and TMB were added sequentially. A450 was determined with an ELISA reader (Ultra 384, Tecan).

NET generation was quantified by examining increased fluorescence emissions from PI in response to DNA binding outside of the cells. Cells were plated in 96‐well plates in the examination buffer. After 2‐4 h of incubation at 37°C, PI was added at 10 mg/mL. A Tecan Ultra 384 fluorescence well plate reader (Männedorf, Switzerland) was utilized to determine fluorescence levels at an excitation wavelength of 360 nm and emission wavelength of 612 nm.

Cell viability was assessed using crystal violet colorimetric assay. Adherent live cells fixed with methanol were stained with 5% w/v crystal violet solution before solubilized with 1% v/v SDS solution. Absorbance at 570 nm was read using a spectrophotometer (Tecan Ultra 384).

Inhibition of gp41 six-helix bundle formation by a testing peptide was determined with a sandwich ELISA described previously57 (link). Briefly, a testing peptide (ADS-J1 as a control) at graded concentrations was preincubated with peptide N36 (1 μM) at 37 °C for 30 min, followed by the addition of peptide C34 (1 μM) and incubation at 37 °C for another 30 min. The mixture was added to a 96-well polystyrene plate (Costar, Corning Inc., Corning, NY) precoated with anti-N36/C34 antibodies (2 μg/ml) purified from mouse antisera specifically against the gp41 six-helix bundle58 (link). Then, mAb NC-1, HRP-labeled rabbit-anti-mouse IgG (Sigma), and TMB were added in order. A450 was determined by an ELISA reader (Ultra 384, Tecan).

The inhibitory activity of peptides on 6-HB formation was measured by a modified ELISA as previously described [48 (link)]. Briefly, a 96-well polystyrene plate (Costar, Corning Inc., Corning, NY, USA) was coated with 50 μL 2 μg/mL NY364 (a polyclonal antibody for HIV-1 gp41 subunit) in 0.1 M Tris buffer (pH 8.8). A mixture of 1 μM N46 and C-peptide with graded concentrations was added to the coated plate after incubation at 37 °C for 30 min. After another 1 hour incubation at 37 °C, the plate was washed with washing buffer (PBS containing 0.1% Tween 20) three times and refilled by 50 μL 1 μg/mL NC-1 (a monoclonal antibody specific for HIV-1 gp41 6-HB formed by NHR and CHR) [49 (link)]. Afterwards, 50 μL of horseradish peroxidase (HRP)-labeled rabbit anti-mouse antibody (Sigma, St. Louis, MO, USA) (1:4000 diluted) were added to the wells of plate, followed by incubation for 1 h and washing. Finally, the substrate 3,3,5,5-tetramethylbenzidine (TMB, Sigma, St. Louis, MO, USA) was added. Absorbance at 450 nm (A450) was tested by an ELISA reader (Ultra 384, Tecan, NC, USA).