Heraeus fresco 17 centrifuge

The content of growth factors was assessed using ELISA [22 (link)]. Briefly, samples were weighed and proteins were extracted using a radioimmunoprecipitation assay extraction buffer (R0278, Sigma-Aldrich, Ireland) with a protease inhibitor cocktail (P9599, Sigma-Aldrich, Ireland). To each sample, 1 mL of extraction buffer was added and samples were incubated in a tissue homogenizer (TissueLyser LT, Qiagen, UK) overnight at 50 rpm and 4 °C. Samples were then centrifuged (Heraeus Fresco 17 Centrifuge, Thermo Fisher, Ireland) at 13,000 rpm and 4 °C for 15 min. Supernatants were then concentrated using Pierce™ 3K Concentrators (Thermo Fisher, Ireland), and basic fibroblast growth factor (FGF-basic), vascular endothelial growth factor (VEGF), and transforming growth factor beta-1 (TGF-β1) content was measured using ELISA DuoSet® kits (DY233, DY293B and DY240, respectively; R&D Systems, UK), as per manufacturer's protocols.

The total and free amino acids were determined as described by Machado et al. [21 (link)]. Briefly, total amino acids of conchocelis were quantified after submitting samples to chemical hydrolysis under alkaline (4 M KOH, 110 °C, 6 h) and acid (6 M HCl, 110 °C, 24 h) conditions. Free amino acids were extracted from the freeze-dried hydrolysate (H-Prolyve and H-ProFla) and the control (unhydrolysed) using solid-liquid extraction in deionised water (1.67% w/v), under repeated cycles of agitation (Multi RS-60, Biosan, Latvia) at room temperature for 45 min. Following centrifugation 17,000× g, for 10 min (Heraeus Fresco 17 centrifuge, Thermo Fisher Scientific, Osterode am Harz, Germany), the supernatants were collected and transferred into injection vials for amino acid analysis. L-Norvaline (2 mg/mL) was used as internal standard. Prior to HPLC analysis, samples were submitted to an automatic pre-column online derivatisation in an AS-4150 Autosampler (Jasco, Tokyo, Japan) using two derivatisation reagents (OPA/3-MPA and FMOC) as described by Machado et al. [21 (link)]. The individual amino acids were identified based on the retention time of known standards and quantified by the internal standard method. Extraction and determination of the total and free amino acid contents were performed in triplicate and the results presented as mg of amino acid/g FDS.

The presence of soluble collagen type I was assessed with sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS-PAGE) [35 (link)]. Briefly, small pieces of each material were cut, weighed, and incubated in 1 mg/mL pepsin (P6687, Sigma-Aldrich, Ireland) in 0.5M acetic acid overnight at 4 °C under continuous agitation (1 mg of material per 1 mL of pepsin/acetic acid solution). Solutions were then centrifuged (Heraeus Fresco 17 Centrifuge, Thermo Fisher, Ireland) at 13,000 rpm and 4 °C for 15 min, supernatants were recovered and loaded onto a Mini-Protean 3 SDS-PAGE unit (Bio-Rad Laboratories, UK). Three percent stacking and 5% separation gels were used. Purified collagen type I (CBP2US, Symatese, France) was used as standard. Gels were stained using the SilverQuest™ Silver Staining kit, as per manufacturer's protocol (Thermo Fisher, Ireland).

Leaf H₂O₂ contents were estimated on the basis of the H₂O₂-induced absorption change in 125 μM xylenol orange in 6% (v/v) trichloroacetic acid (TCA), as described previously [15 (link)]. In order to avoid undesirable rapid changes in steady-state leaf H₂O₂ levels, leaf disks for this measurement were cut while plants were still in the growth chamber under light conditions corresponding to the treatment: PAR only or PAR plus UV-B. Three leaf disks corresponding to 26–56 mg FW were homogenized in 6% TCA immediately after cutting and then centrifuged (15,000× g, 10 min, 4 °C, Heraeus Fresco 17 Centrifuge, Thermo Fisher Scientific, Waltham, USA), and the supernatants were incubated for 30 min before detecting 560 nm absorptions. Leaf H₂O₂ contents were calculated in μmol g⁻¹ FW units using calibration in the 0–10 nM H₂O₂ range.

Samples were mixed in a vortex mixer before being centrifuged in a Heraeus™ Fresco 17 centrifuge (Thermo Fisher scientific, MA, USA) at 12,000 × g for 10 min. The supernatant was analysed using a rapid and sensitive HPLC assay derived from methods described by Couto et al. (27 ) to detect and quantify fluticasone propionate on inhalation particles. A 1260 Infinity II LC System (Agilent, CA, USA) was coupled with isocratic elution on an Extend-C₁₈ column using acetonitrile and water (80:20, v/v) with the flow rate set at 0.5 mL/min. The UV detector was set to 236 nm and the total run time was 5 min. Calibration curves were prepared to assess linearity, precision, and accuracy (Fig. 7). Precision and accuracy were deemed acceptable if measured values were within ± 15% of the actual values (± 20% at the lower limit of quantification, LLOQ). Good linearity (r² = 0.99) was obtained in the range of 0.02 to 0.40 mg/mL for fluticasone propionate.
Fig. 7

Standard curve obtained to quantify fluticasone propionate

Leaf H₂O₂ levels were estimated using a photometric assay⁷⁹ based on the H₂O₂-induced absorption change of 125 μM xylenol orange in 6% (v/v) trichloroacetic acid (TCA). For this assay, samples were collected from plants within the growth chamber under light conditions corresponding to treatment groups i.e. PAR only or PAR plus UV-B. Three leaf disks corresponding to 26–56 mg FW were homogenised in 6% TCA immediately after cutting, centrifuged (15,000×g, 10 min, 4 °C, Heraeus Fresco 17 Centrifuge, Thermo Fisher Scientific, Waltham, USA), and the supernatants were incubated for 30 min before detecting 560 nm absorptions. Leaf H₂O₂ contents were given in nM mg⁻¹ FW units using calibration curves in the 0–10 nM H₂O₂ range.

Poly(hydroxymethyl 3,4-ethylenedioxythiophene) hollow microspheres (MSP) were fabricated using a sacrificial template chemical polymerisation process^{36 (link)}. In brief, EDOT-OH was dissociated in a sonication bath at a frequency of 35 kHz for 30 min. Following this, polystyrene (PS) beads with an average diameter of 500 nm (Spherotech inc., CHI, USA) were added to the EDOT-OH solution to a final concentration of 2.5 mg ml⁻¹. The mixture was allowed to homogenise using a magnetic stirrer for 30 min before ammonium persulphate was added to initiate polymerisation of EDOT-OH around PS bead templates. The solution was then mixed for 16 h at room temperature before the addition of excess methanol ceased the reaction. Following this, the solution was centrifuged (Thermo Scientific HERAEUS FRESCO 17 Centrifuge) at 13,000 rpm for 15 min to remove excess neutralised solution, and washed three times with deionized water by resuspension and repetitive centrifugation. To dissolve the internal polystyrene template, MSP were suspended in tetrahydrofuran, THF (Sigma Aldrich) for 15 min followed by repetitive washing in deionized water as described earlier.