Alliance hplc system

Mitochondria were isolated from cultured cell lines as previously described [21 (link)] with small modifications [22 (link)]. A mitochondrial pellet was immediately frozen in liquid nitrogen under anaerobic conditions. Then, lipids extraction and CoQ determination were performed as described previously [23 (link)]. Briefly, mitochondrial samples (0.6 mg of mitochondrial protein for each measurement) were lysed with 1% SDS and vortexed for 1 min. Then, a mixture of ethanol:2-propanol (95:5) was added and the samples vortexed again for 1 min. To recover CoQ, 5 mL of hexane was added, and the samples were centrifuged at 1000× g for 5 min at 4 °C. The upper phases from two extractions were recovered and dried using a rotary evaporator. Lipid extracts were suspended in 1 mL ethanol, dried in a speed-vac and stored at −20 °C. Samples were resuspended in a suitable volume of ethanol prior to HPLC injection. Lipid components were separated with an Alliance HPLC system (Waters) equipped with a 2707 autosampler and 2996 photodiode array detector, with HSST3 column (4.6 × 150 mm, 3.5 µm, Waters), preceded by a pre-column (4.6 × 20 mm, 3.5 µm, Waters), with a flow rate of 1 mL/min and a mobile phase containing 40:60 methanol/2-propanol. Commercial CoQ10 (Sigma) was used as an internal standard to detect the CoQ10 peak in the samples.

To assess the extent of acetaminophen metabolism, urine was collected over 24 h post exposure of the ¹⁴C-acetaminophen dose and immediately frozen at −20 °C. Prior to HPLC analysis, each urine sample was thawed, and a 0.1–0.5 mL aliquot from each fraction was analyzed by liquid scintillation counting to determine the total 14-carbon content. Each sample was then analyzed by reversed-phase HPLC for acetaminophen and acetaminophen metabolites. After centrifugation of approximately 150 µL of each urine sample at 10,000 RPM for 5 min, 100 µl of the supernatant was directly injected into an Alliance HPLC system (Waters, Milford, MA) equipped with a 4 μm, 4.6 × 250 mm Synergi Max-RP 80 A column (Phenomenex, Torrance, CA), and monitored at 248 nm. Metabolites were eluted at 1.0 ml/min using a solvent of 88% 0.1 M KH₂PO₄ containing 0.75% glacial acetic acid and 12% methanol. The column eluent was collected at 1 min intervals, and radioactivity was quantified by liquid scintillation counting.
Acetaminophen glucuronide and sulfate conjugates were determined based on their susceptibility to β-glucuronidase and sulfatase, respectively^{30 (link)}.

To evaluate the HSL lytic activity of the purified AiiM, an analytical assay was developed in an Alliance HPLC system (Waters, United States) with a Symmetry (Waters, United States) C18 Column (75 mm, 3.5 mm). Both short and long acylated chains were included, 1 mM N-butyryl-DL-homoserine lactone (C4-HSL; Sigma Aldrich 09945), 1 mM N-(3-oxooctanoyl)-L-homoserine lactone (3OC8-HSL;Sigma Aldrich O1764), 1 mM N-decanoyl-DL-homoserine lactone (C10-HSL; Sigma Aldrich 17248), 1 mM N-(3-oxodecanoyl)-L-homoserine lactone (3OC10-HSL; Sigma Aldrich O9014), and N-(3-oxododecanoyl) homoserine lactone (3OC12-HSL; Sigma Aldrich O9139). 60 mM NaOH was used as positive control since it can hydrolyze HSL molecules and the reaction was stopped with the addition of 2N HCl. Several concentrations, from 250, 100, 50, 25, 10, and 5 μg/mL of purified AiiM were tested. Time exposition was also varied; 24 h, 2 h, 1 h, 30 min, 20 min, 10 min, and 5 min. All experiments were performed in triplicate. Chromatographic conditions were: column temperature 25°C, sample temperature 25°C, injection volume 10 μL, flow rate 1 mL/min, detection 205 nm. Elution mixture was made with 50 mM phosphates buffer pH 2.9:acetonitrile. For C4-HSL the relation was 90:10, 3OC8-HSL 60:40, C10-HSL 60:40, 3OC10-HSL 60:40, and 3OC12-HSL 50:50.

A 5 µL aliquot of WAC-288 spore stock was sub-cultured on MYM agar for 7 days. Colonies were selected and grown in 4 × 700 mL liquid R5 media (−50% maltose)⁶⁹ shaking at 200 rpm in baffled flasks for 7 days. Cultures were sonicated for 10 min and centrifuged at 20,000 × g to remove the cell pellet. Spent media was filtered and continuously mixed with 20 g of Diaion® HP-20 Resin overnight. Resin was packed into an empty column cartridge and flash chromatography was performed using the Reverleris X2 system (Grace). Reverse-phase HPLC was carried out with a 250 ×4.6 mm C18 5 μm column (Phenomenex) on the Alliance HPLC system (Waters). LC-MS/MS data were obtained using an Acquity UPLC (Waters) with an inline/Xevo G2-S qTOF (Waters) and processed with MassLynx V4.1 and MZmine 2 Software. Please refer to supplementary methods for complete cosmomycin isolation details. Small-scale larval assays to detect bioactivity were carried out in a microcentrifuge cap with 15 μL dH₂O and 5 μL suspended aliquots of flash and HPLC fractions. Twenty-first instar larvae were placed in the droplet and caps were covered in parafilm for 2 days.

An accelerated aggregation study of mAb2 WT and the variant mAb2_SL50K_SH21R_SH85R was performed. Samples of 5 mg/mL in 25 mM sodium citrate and 125 mM sodium chloride (pH 6.0) were incubated at 40 °C over a time span of 6 months (180 days). The unstressed samples (t = 0) were analyzed immediately after preparation. All 40 °C incubated samples were stored at − 70 °C and analyzed together at the end of the study. The amounts of aggregation and remaining monomer content were determined by SEC on an Alliance HPLC system (Waters, Milford, MA, USA) employing a TSKgel G3000 SWX column (Tosoh Bioscience LLC). The monomer loss overtime was calculated as slope (m) of the line defined by the monomer content of a sample at the initial and end time point of the study.
The mobile phase was 50 mM Tris/HCl and 150 mM sodium chloride buffer (pH 7.5) at a flow rate of 1 mL/min. Areas of peaks followed by 280 nm are integrated at each time point. All samples were measured in duplicates.