96 well filter plate

The concentration of TNF-α, IFN-γ, IL-1α, IL-5, IL-10, IL-12p70, IL-17, and eotaxin was analyzed by cytometric bead array using the manufacturer's specifications (BD Biosciences, USA). Briefly, starting with cytokine standard preparation and cytokine capture bead mixture, 50 μl serum samples from each individual and dilutions for the standard curve were placed in a multiscreen 1.2 μm, 96-well filter plate (Merck Millipore, Germany), and the mixed capture beads and phycoerythrin detection reagent were added. After 3-hour incubation and repeated wash steps, the data was acquired in a FACSCanto II flow cytometer (Becton Dickinson, USA) and analyzed using FCAP Array v3.0 (BD Biosciences, USA) to convert fluorescent intensity values to concentrations using a standard curve.

Thermostability assays were performed using a modified version of previously described methods (Lebon et al., 2011a (link); Serrano-Vega et al., 2008 (link)). Insect cell membranes containing β₁AR^∆NC were resuspended in assay buffer (25 mM HEPES pH 7.5, 400 mM NaCl, 1 mM MgCl₂, 1 mM ascorbate, 0.1% BSA, 0.004% bacitracin). The sample was aliquoted and binding partner (25 μM final concentration), ³H-norepinephrine (200 nM final concentration) and apyrase (0.1 U/ml final concentration) were added. Norepinephrine was added to the negative control (200 μM final concentration). Samples were incubated at 4°C for 1 h, before solubilisation with detergent for 1 h on ice. The detergents dodecyl maltoside (DDM), decyl maltoside (DM), nonyl glucoside (NG) or octyl glucoside (OG) were used at final concentrations of 0.1, 0.13, 0.3 or 0.8%, respectively. Samples were heated to different temperatures (between 4 and 50°C) for exactly 30 min, followed by quenching on ice for 30 min. Samples were separated by gel filtration through Toyopearl HW-40F resin packed in a 96-well filter plate (Merck Millipore). Radioactivity was quantified by scintillation counting and apparent melting temperature (T_m) values were determined using GraphPad Prism version 5.0.

A solid/liquid lipid extraction method, previously described in detail in Lauer et al. (2021), was used to detect lipids [25 (link)]. A 96-well filter plate (0.45 μm; Merck, Darmstadt, Germany) was attached to a 96-deep-well plate (Fisher Scientific, Schwerte, Germany), and circles of Whatman blotting paper with a diameter of 6 mm were added to each well. Then, both a standard mixture and 10 µL of the prepared sample were added to each Whatman paper. The samples were dried for 45 min under a nitrogen flow (1–2 bar) and then 20 μL of 5% PITC (v/v) diluted in ethanol/water/pyridine (1:1:1, v/v/v) was added to the samples. Before drying again for 45 min under nitrogen, the samples were incubated for 20 min at room temperature. After drying, lipids were extracted using 300 μL 4.93 mM ammonium acetate in methanol, and the plate was shaken for 30 min at 450 rpm on a plate shaker (IKA, Staufen, Germany). Centrifugation for 2 min at 500× g transferred the liquid samples to the 96-well plate, followed by dilution of the samples with 600 μL 5 mM ammonium acetate in methanol/water (97:3, v/v). After covering the plate with a silicone mat, they were shaken at 450 rpm for 2 min at room temperature and then analyzed by mass spectrometry.

Reaction mixtures with 0.2% (w/v) PASC, 1 μM of AtAA9 and 1 mM ascorbic acid were set up in 800 μL total volumes in 50 mM of BisTris-HCl pH 6.0 and incubated as specified above. Samples (150 μl) were collected after 20, 40, 60, 120, and 240 min of incubation and boiled at 97°C for 10 min to stop the reaction. Soluble and insoluble fractions were separated using a 96-well filter plate (Merck Millipore) and a Merck Millipore vacuum manifold. Next, 25 μl of the soluble fractions were supplemented with 1 μL TrCel7A in 150 mM Na-acetate pH 4.75 (to a final concentration of 1 μM), followed by incubation at 37°C for 18 h in order to convert the solubilized oxidized oligosaccharides to the corresponding oxidized dimers. After the incubation, the samples were incubated at 97°C for 10 min to stop the reaction. For product quantification, cellobionic acid (as C1-oxidized) and C4-oxidized dimer standards were prepared as described before [5 (link), 52 (link)].

Following humane sacrifice, alveolar macrophages were harvested from gp91^-/- and WT mice by broncho-alveolar lavage (BAL). Lavage was performed four times using 1 ml ice cold PBS supplemented with 5 mM ultrapure ethylenediaminetetraacetic acid (EDTA, Invitrogen Life Technologies) via an 18 gauge (G) intravenous cannula (Introcan-W, Braun) inserted into the trachea. Samples were centrifuged at 500 x g for 5 min at 4°C, supernatants discarded, and the resulting cell pellet re-suspended in 300 μl RPMI supplemented with 10% HI FBS. Cells from a minimum of six mice per group were pooled, counted, and their viability checked using Trypan Blue.
Cell suspensions were adjusted to between 1 x 10⁵ and 5 x 10⁵ cells/ml and 100 μl of the resulting cell suspension transferred to a 96-well filter plate (Merck Millipore). Plates were then incubated for three hours at 37°C and 5% CO₂ to allow the macrophages to adhere. After three hours the media was refreshed before adding conidia to the wells at a ratio of 5 conidia to 1 AM. Plates were then incubated for 12 or 18 hours at 37°C and 5% CO₂ before fungal viability was assessed using a colorimetric XTT assay as described by Henriet et al. (Henriet et al., 2011 (link))

To assess the impact of H₂O₂ on product generation by ScLPMO9A acting on PASC, reactions containing 1 µM LPMO, 2 g/L PASC, 1 mM AscA, and 0, 50, 100, or 250 µM H₂O₂ in 50 mM Tris–HCl pH 7.5 were prepared. The reactions were initiated by addition of AscA and incubated at 45 °C and 1000 rpm in a Thermomixer (Eppendorf). H₂O₂ was added to the reactions immediately prior to the AscA. Samples were taken at 3, 6, 9, 30, and 60 min, and remaining insoluble substrate was removed by filtration using a 96-well filter plate (0.45 µm; Merck Millipore) operated with a Millipore vacuum manifold system. Samples were subsequently stored at – 20 °C prior to analysis by HPAEC-PAD. All reactions were performed in triplicate, and control reactions without addition of AscA were performed in parallel.
Reactions with cellopentaose contained 1 µM LPMO, 1 mM cellopentaose, 50 µM AscA, and 200 or 400 µM H₂O₂ in 50 mM sodium acetate pH 5.0. Immediately following the addition of H₂O₂, reactions were initiated by addition of AscA and incubated as described above. Samples were taken at various time points and reactions were quenched by addition of NaOH to a final concentration of 100 mM. Samples were subsequently stored at – 20 °C prior to analysis of generated native products by HPAEC-PAD. All reactions were performed in triplicate, and control reactions without addition of AscA were performed in parallel.