Hplc vials

CIP was purchased from Sigma Aldrich (St. Louis, MO, USA). Labrafac^® Lipophile WL 1349 (LWL) was a generous gift sample from Gattefossé (Paramus, NJ, USA). Oleic acid (OA), Tween^® 80, Poloxamer 188, and 0.5 mL cup-like design Thermo Scientific™ Slide-A-Lyzer™ MINI Dialysis Device (10 K molecular weight cutoff) were obtained from Fischer Scientific (Hampton, NH, USA). Other chemicals and glassware required for the project such as high performance liquid chromatography (HPLC) grade solvents, scintillation vials, centrifuge tubes, HPLC vials were acquired from Fischer Scientific (Hampton, NH, USA) and Gattefossé (Paramus, NJ, USA). Whole eyes of male albino New Zealand rabbits were purchased from Pel-Freez Biologicals (Rogers, AR).

Isolates of Aspergillus section Flavi were cultured into semi-solid (slant), solid and submerged conditions. For the semi-solid (slant) culture, 2 mL of Potato Dextrose Broth (PDB, Difco Lab, Sparks, MD, USA) was added in 25 mL glass test tubes, and fungal isolates (5 × 10⁵ conidia/tube) were inoculated with the help of an inoculating loop. The tubes were put in a rack at a 45°angle and placed in an incubator at 28 ± 2 °C for seven days. For solid culture, fungal isolates (5 × 10⁵ conidia/petri dish) were inoculated in petri dishes containing 25 mL of PDA (Difco Lab, Sparks, MD, USA) and placed in an incubator at 28 ± 2 °C for seven days. For submerged culture, fungal isolates at 5 × 10⁵ conidia/flask were inoculated in 250 mL Erlenmeyer flasks containing 100 mL of PDB. All the flasks were incubated for seven days at 28 ± 2 °C with shaking at 220 rpm. After 7 days of incubation, aflatoxins were extracted from semi-solid, solid, and submerged cultures of fungal isolates as described in Alshannaq et al. [88 (link)]. Prior to HPLC analysis, all samples were filtered (0.45 mm with a diameter of 47 mm) (Thermo Fisher Science, Rockwood, TN, USA) into HPLC vials via a PTFE 0.45 μm syringe filter (0.45 mm with a diameter of 17 mm) (Thermo Fisher Science, Rockwood, TN, USA).

When sufficient cells were available from donors, a rapid assessment of M8OI metabolism was performed using high concentrations of M8OI and an HPLC with UV detection. The higher concentrations used enabled the metabolic capacity to be determined using this less sensitive methodology, which was routinely available for rapid determinations. This rapid turnaround also enabled decisions to be made regarding experiments with cells, which remain differentiated for a short time only (Wallace et al., 2010 (link)). Freshly isolated hepatocytes were cultured overnight to allow for attachment, washed and cultured in STIM buffer supplemented with M8OI. Samples of this incubation were taken at various times and 1 vol of 1 % HPLC-grade phosphoric acid was added to 10 volumes of sample. The sample was vortexed and centrifuged for 1 min at 16,000 g. The supernatant was transferred to HPLC vials (Thermo Scientific) and subjected to HPLC using an LC-20AD system with Nucleosil C18 column (25 cm x3.2 mm). Samples were eluted with a mobile phase gradient system of (A) 0.1 % phosphoric acid (Fisher) and (B) acetonitrile (Fisher) at a flow rate of 0.7 mL/min [0 min: A = 90 % / B = 10 %; 15 min: A = 10 % / B = 90 %; 30 min: A = 10 % / B = 90 % and 35 min: A = 90 % / B = 10 %]. M8OI and COOH7IM were detected at 211 nm and concentrations were determined using authentic standard.

A total of three different NRRV P[4] fusion-protein antigens were used in these studies including E. coli expressed parent protein P[4], K. phaffii (P. pastoris) expressed parent protein P[4], and K. phaffii (P. pastoris) expressed P[4] variant (C173S), which are hereafter referred as E. coli P[4], Pp P[4] and Pp P[4]-C173S, respectively. The detailed description of the design of these P[4] mutants as well as the procedures for their expression and purification are described elsewhere^{31 ,32} (link) and in the companion paper.²³ The purity of these three P[4] samples was estimated to be >90% as determined by SDS-PAGE analysis and densitometry (Supplementary Fig. S1A). Sodium phosphate dibasic heptahydrate and sodium chloride (NaCl) were purchased from Fisher Chemicals (Hampton, NH). Sodium phosphate monobasic monohydrate, 8-anilino-1-naphthalenesulfonic acid (ANS), thimerosal (TH), and dimethyl sulfoxide (DMSO) were purchased from Sigma-Aldrich (St. Louis, MO). Slide-A-Lyzer mini dialysis devices, HPLC vials, LC-MS grade mobile phases and isopropanol were purchased from Thermo Fisher Scientific (Waltham, MA). NRRV P[4] specific mAb was developed by PATH and obtained from Precision Antibody (Columbia, MD) as described elsewhere.²² (link)^,33 (link)