Thermomixer c

For maintenance, S.
pombe strains were cultivated in YES or EMM2 solid
media at 28 °C.
For general preculture, S. pombe strains were cultivated in YES or EMM2 at
28 °C and 200 rpm in an orbital shaker.
For experiments
measuring the promoter and terminator strength, S.
pombe strains were cultivated in 1 mL volumes
in 96-deep-well plates (CR1496, Enzyscreen) sealed with AeraSeal (Excel
Scientific), at 28 °C and 1500 rpm on an Eppendorf ThermoMixer
C (Eppendorf).
For metabolic pathway expression experiments, S.
pombe strains were cultivated in EMM2 in 24-deep-well
plates (CR1426, Enzyscreen) sealed with AeraSeal (Excel Scientific),
at 28 °C and 800 rpm on an Eppendorf ThermoMixer C (Eppendorf).
For plasmid amplification, E. coli strains were cultivated in LB medium in 24-deep-well plates (CR1426,
Enzyscreen) sealed with AeraSeal (Excel Scientific), at 37 °C
and 800 rpm on an Eppendorf ThermoMixer C (Eppendorf).

Paclitaxel (PTX) loading into pEV was performed as previously described by Kim et al., with minor modifications [35 (link)]. In brief, 50 μM (64 μg/mL) of PTX or DMSO vehicle (Dimethyl sulfoxide, CryoSure-DMSO, WAK-Chemie Medical GmbH) were mixed with 5 × 10¹⁰ pEV in DPBS. Samples were incubated for 1 h at 37 °C with constant agitation at 350 rpm (ThermoMixer^® C Eppendorf). Free drug was removed using DGUC (Section 2.3.1). The resulting PTX-pEV samples were used immediately for characterization or functional experiments, or stored at 4 °C until further use.
A calibration curve (Supplementary Figure S1) for the indirect quantification of PTX loaded into pEV was prepared according to previous publications [36 ,37 (link)]. The PTX stock solution was diluted with 30% (v/v) methanol in DPBS to achieve concentrations between 2.0–20.0 μg/mL. The calibration values were measured by UV at a wavelength of 230 nm (Shimadzu UV-1603 spectrophotometer) with correction for the blank. Once PTX was dissolved in DMSO, the absorbance values were corrected against the corresponding values in DMSO.
To measure PTX concentration in pEV, approximately 2 × 10⁹ PTX-pEV were treated with 100 μL of 1 × RIPA buffer (Sigma) and placed on a thermomixer (ThermoMixer^® C Eppendorf) for 30 min at 4 °C with shaking (650 rpm). Subsequently, the absorbance was measured as previously described [38 (link)].

Approximately 150 mg of the real sample or the pea-based matrix sample was placed in a 2-mL Eppendorf tube; 1 mL of a 20% solution of sodium chloride and 300 µL of DES were added (Fig. 1). The sample was mixed for 1 min (1700 rpm, ThermoMixer C, Eppendorf, Hamburg, Germany) and centrifuged for 5 min (6000 rpm, Centrifuge 5084 R, Eppendorf, Hamburg, Germany) to separate the phases. At this stage, the DES phase was coalesced at the top of the vial and was easily withdrawn and transferred into a clean 1.5-mL Eppendorf tube containing 0.5 mL of 5% formic acid solution. Then, the back-extraction step was performed, and the mixture was mixed for 1 min (1700 rpm, ThermoMixer C, Eppendorf, Hamburg, Germany) and centrifuged for 5 min (6000 rpm). At this stage, due to lower density of the formic acid solution in comparison to the organic phase, the coalesced phase inversion was observed, and DES was separated at the bottom of the tube. The aqueous phase (upper one) was collected and filtered through syringe filter (0.22 µm, ø 13 mm, nylon) and placed in the autosampler vial to be analysed by LC-MS/MS. The procedure was applied to real samples, fortified pea-based samples and matrix-matched calibration solutions.Fig. 1

The schematic representation of the hydrophobic natural deep eutectic solvent-based microextraction procedure for the analysis of plant-based meat substitutes