Millex filters

DLS characterization of PTX/niosome was carried out using a dynamic light scattering (DLS) (Malvern, Helix, UK) system coupled with a diode-pumped solid-state laser to supply polarized incident light. A digital correlator with a sample range of 25 ns to 100 ms was also included in the device. DLS was conducted by calibrating the intensity scale with toluene against scattering at an angle of 90° to the incident ray. The sample solutions were filtered directly into scattering cells using Millipore Millex filters (0.22 m porosity) and equilibrated for 10 min at the appropriate temperature before being measured. The sampling time was 5–10 min to obtain a fitted correlation function. All of the tests were repeated three times.

ABA, IAA, SA, JA and JA-Ile were analysed in the sap collected for Lo measurement according to Albacete et al. [93 (link)] with some modifications. Briefly, xylem sap samples were filtered through 13 mm diameter Millex filters with 0.22 µm pore size nylon membrane (Millipore, Bedford, MA, USA). The deuterium-labelled internal standard used for hormones determination were the following: ²H₅-Indole-3-Acetic acid (D-IAA), ²H₆-(+)-cis,trans-Abscisic acid (D-ABA), ²H₂-N-(-)-Jasmonoyl Isoleucine (D-JA-Ile) and ²H₄-Salicylic acid (D-SA), obtained from OlChemin Ltd. (Olomouc, Czech Republic). The ²H₅-Jasmonic acid (D-JA) was obtained from CDN Isotopes (Pointe-Claire, QC, Canada). Ten µL of each internal standard was added to the filtrate. Subsequently, 10 µL of filtrate were injected in a U-HPLC-MS system consisting of an Accela U-HPLC system (ThermoFisher Scientific, Waltham, MA, USA) coupled to an Exactive^TM mass spectrometer (ThermoFisher Scientific) using a heated electrospray ionization (HESI) interface. Mass spectra were obtained using Xcalibur software version 2.2 (ThermoFisher Scientific, Waltham, MA, USA). For quantification of the plant hormones, calibration curves were constructed for each analysed component (1, 10, 50, and 100 µg L⁻¹).

Unless otherwise stated, reagents and solvents were commercially available and used without further purification: N-Boc-protected nitro-precursor (Part No. NPPI-95-0010C) and authentic AV1451 reference standard (Part No. FPPI-95-0002A) were purchased from Huayi Isotopes/NucMedCor. Ethanol (200 proof, USP) was purchased from Decon Laboratories, Inc. Sodium chloride 0.9%, USP and sterile water for injection, USP were sourced from Hospira. Other synthesis components were obtained as follows: Sterile vials were obtained from Hollister-Stier; Millex filters were from Millipore; QMA-light and Oasis HLB 1 cc cartridges were purchased from Waters. Prior to use QMA cartridges were conditioned with ethanol (10 mL), 0.5 M NaHCO₃ (10 mL) and sterile water (10 mL), while HLB cartridges were conditioned with ethanol (10 mL) and sterile water (10 mL).

Lonomia obliqua crude bristle extract (LOCBE) was obtained from caterpillars collected in the south of Brazil (states of Santa Catarina, Rio Grande do Sul and Paraná) and provided by the Butantan Institute. Briefly, to obtain the extract, caterpillar’s bristles were harvested by cutting them at their base, ground in a mortar and homogenized in PBS (pH 7.4). The solution was sterilized by filtration using Millex filters (Millipore, Darmstadt, Germany, #SLGV013SL). Protein content was evaluated using the Pierce BCA Protein Assay Kit (Thermo Scientific, Rockford, IL, USA), according to the manufacturer’s protocol, and stored at −80 °C until use.
The endotoxin levels present in LOCBE samples were assessed following Good Manufacturing Practice (GMP) using the Gel Clot—limit test assay. Endotoxin was detected at an acceptable level between 0.1 and 1.2 EU/mL at 5 μg/mL LOCBE. LPS 1 μg/mL (99% purity) was employed as a positive control.

The Targas-1 equipment (PP Systems, Amesbury, MA, USA) was used to determine photosynthetic traits, including the leaf internal concentration of CO₂ (Ci: μmol mol⁻¹), photosynthesis rate (A: μmol CO₂ m⁻² s⁻¹), and stomatal conductance (gs: mmol H₂O m⁻² s⁻¹), following the instructions provided in the user manual. Measurements were conducted on the second youngest leaf from four plants per treatment, one day before harvest. To extract photosynthetic pigments, 0.5 g of fresh material was mixed with 5 mL of methanol for 24 h. The resulting samples were then filtered using 13 mm diameter Millex filters with a 0.22 μm pore size nylon membrane (Millipore, Bedford, MA, USA). The absorbance of the filtered liquid was measured at wavelengths of 663 nm and 645 nm, corresponding to chlorophyll A and B, respectively, using a Synergy H1 Hybrid Multi-Mode microplate spectrophotometer (BioTek Inc., Winooski, VT, USA). The concentrations of pigments were calculated using the provided formula by Lichtenthaler [106 ].

We have previously shown that Dulbecco's modified essential medium (DMEM) low glucose (5.6 mM) medium (Invitrogen) is the optimal growth medium for inducing K279a protease activity (20 (link)). To prepare a stock solution of K279a CS, 10 μL of an overnight K279a culture was inoculated in 6 x 15 mls of DMEM low glucose (5.6 mM) medium and grown for 48 h at 37°C on an orbital shaker. K279a CS was passed sequentially through 0.45-μm and 0.2-μm filters millex filters (Millipore Corporation, Bedford, MA). Culture supernatant (90 mls) was then concentrated using 5-kDa nominal-weight limit (NMWL) cut-off Amicon® Ultra-15 filter devices (Millipore Corporation, Bedford, MA). All concentrates were centrifuged at 4,000 × g and subsequently diafiltered by centrifugation with sterile DPBS to remove any low molecular weight contaminants including glucose and amino acids present in DMEM. An equivalent volume of DMEM was used as a negative control and for correction during protein quantification using the BCA (bicinchoninic acid) assay.