Infinite m200 pro microplate reader

Solutions
of the 50
μM tau^4RD protein in 20 mM sodium phosphate buffer
at pH 7.4, 50 mM NaCl, 1 mM DTT, 0.02% NaN₃, and protease
inhibitors with EDTA were incubated in the absence or presence of
coffee extract or different compounds in 96-well dark plates at 37
°C for 40 h. Heparin and Thioflavin-T (ThT) were added to the
sample solutions in a molar ratio of 1:1 with respect to the protein.
Fluorescence measurements (λex.: 450 nm and λem: 482 nm) were performed with a Tecan Infinite
M200 Pro Microplate Reader (Tecan Group AG, Männedorf, Switzerland)
with cycles of 30 s of orbital shaking at 140 rpm and 10 min of rest
before the fluorescence reading throughout the incubation, as described
in previous work.^{15 (link)} The fluorescence intensity
and lag-phase duration of four replicates of each sample were analyzed
with GraphPad Prism 8.2 software (GraphPad Software, San Diego, California, www.graphpad.com). Any pre-existing
aggregate was removed by filtering the protein stock solutions through
a 100 MWCO cut-off filter (Sartorius Stedim Biotech GmbH, Göttingen,
Germany), before the aggregation reaction. Error bars of ThT curves
correspond to standard deviations of four independent experiments.

Biofilm production was evaluated using the crystal violet staining assay described by O’Toole and Kolter as described before (O'Toole and Kolter, 1998 (link)) with slight modifications. Briefly, A. baumannii overnight cultures were adjusted to a 0.5 McFarland turbidity in 0.85% saline solution. Biofilms were developed in 24-well flat-bottom plates (Sarstedt^®, Nümbrecht, Germany). First, bacterial suspensions were incubated at 37°C for 24 h. Then, biofilms were washed, air-dried and stained with 1 mL/well of 0.7% crystal violet solution (Sigma-Aldrich). Finally, stained biofilms were solubilized with 1mL/well of 33% acetic acid solution (Sigma-Aldrich).Biofilm production was determined at 600 nm using the Tecan Infinite M200 Pro Microplate Reader (Tecan Group Ltd., Männedorf, Suiza). Results were corrected for background staining by subtracting the value for crystal violet bound to uninoculated Müller Hinton Broth control wells. Isolates E. coli J53 and P. aeruginosa PAO1 were used as negative and positive controls, respectively. The experiments were performed in triplicate and repeated in three different days with similar results.

Briefly, freshly isolated healthy neutrophils were resuspended at 6 × 10⁶ cell/mL in complete RPMI medium supplemented with 25% of cf-MPE-LAC or cf-PE-HF. We also cultured cells with completed RPMI medium as a positive control for NETosis. Then, neutrophils were seeded into a black 96-well plate and stimulated with 25nM phorbol-12-mystrate-13-acetate (PMA; Sigma-Aldrich) for three hours in a humidified incubator (37 °C) or remained unstimulated as a control. Next, cells were collected by centrifugation and supernatant was stained with Sytox^TM Green nucleic acid stain (5 µM; Invitrogen, Eugene, OR, USA) for 20 min. The relative fluorescence was then read with a fluorometer Infinite M200 Pro Microplate reader (Tecan Group, Männedorf, Switzerland) with a filter setting of 485 nm (excitation)/520 nm (emission). Fluorescence intensity reflects the amount of DNA. Results were expressed as relative fluorescence units (RFUs). Endogenous pleural DNA was measured by basal pleural emission (without cells) and subtracted from pleural fluid culture wells.