Bovine serum albumin (bsa)

For northern blots, 600 ng of RNA extracted from the RNase R immunoprecipitation (see above) and 420 ng of total RNA were loaded on 6% TBE-Urea gel (Invitrogen). The gel was run for 1.5 h at 180 V in 1× TBE (Tris-Borate-EDTA) buffer (Thermo Fisher Scientific), after which the blot was conducted on an Amersham Hybond-N+ membrane (Cytiva) in a wet-blot transfer chamber (Bio-Rad) with 0.5× TBE buffer overnight at 40 V (4 °C). The membrane was then dried at 65 °C for 10 min and cross-linked in a Stratagene UV cross-linker (twice at automode). After blocking at 28 °C for 1.5 h in 250 mM Na₂HPO₄ pH 7.2, 1 mM EDTA, 7% SDS, 0.5% BSA (Applichem) and 80 μg ml⁻¹ salmon sperm DNA (Sigma) buffer, 0.5 pmols 5′-Cy3-labelled ssDNA probe (Metabion) was added and the membrane was incubated overnight on a turning device at 28 °C. After washing twice with 2× SSC buffer and 0.2% SDS, and twice with 1× SSC buffer and 0.1% SDS, the blot was visualized using an Amersham Typhoon scanner (GE, Cytiva).The sequences of all probes are listed in Supplementary Table 1.

In a final in vivo experiment, the triglyceride absorption kinetics following ingestion of complete formulas was determined. A total of 49 preterm piglets from three pregnant sows were born by cesarean section at day 106 of gestation. They were stabilized and received parenteral nutrition as described above and were block-randomized according to birthweight into three groups, all receiving the complete formula, with a fat content of 10%. Collectively for all three in vivo studies, we ensured to have pigs from each litter equally represented in all treatment groups, allowing us to correct for any variance between litters and their specific genotype, in the analysis of variance. The complete formulas were made with the emulsions based on SL, WPC-PL, or WPC-A-EV. To stimulate the intestinal absorptive function, we initially fed the pure base-diet without emulsions during the first 24 h, at a rate of 3 mL/kg every three hours. On day two, the complete formulas including the emulsions were given at a bolus dose of 9 mL/kg, following a 6 hr fasting period. Blood samples were collected via the umbilical catheter at t = 0, 30, 60, 90 min after the bolus. Following this, the piglets were again fed with increasing amounts of enteral diet (6–9 mL/kg every three hours). On day four, the piglets were again fed a test meal of 9 mL/kg after a fasting period of 3.5 h, and blood samples were collected at t = 0, 30, 60, 90, and 120 min. Blood was drawn via the jugular vein in cases where the umbilical catheter was dysfunctional. Plasma was isolated, and triglyceride concentration was measured.
The pigs were euthanized on day four following a standardized feeding regimen to ensure an equal amount of gastric content at the time of euthanasia. Specifications for recordings and sample collection are provided in the supplementary section. Gastric content was weighed, and gastric lipase activity in gastric content was measured using the method described earlier with slight modifications [36 (link)]. The assay was carried out using the same pH Stat equipment as for the in vitro lipolysis. It was initiated by mixing 14.5 mL assay medium containing 1.5 µM Bovine Serum Albumin (AppliChem, Darmstadt, Germany), 150 mM NaCl (VWR), and 2 mM sodium taurodeoxycholate (Sigma Aldrich) with 0.5 mL tributyrin (Sigma Aldrich) and 0.5 mL stomach content. The enzymatic activity, calculated as U/mL was based on the measured titration rate of NaOH by a Metrohm Titrando pH Stat over five minutes of digestion at pH 5.5. As the butyric acid was not being fully titratable at pH 5.5, a correction factor of 1.12 was multiplied to the calculated activity. The total amount of collected stomach content, as well as the pH, was also measured.

To obtain a time-course analysis of stimulation with PMA and calcium ionophore Ibidi µ-slides (8-well, Ibidi, Martinsried, Germany) were coated with a 1:5 dilution of Poly-L-Lysine (Sigma-Aldrich), washed once with sterile water and air-dried. Neutrophils of 4 donors were isolated and seeded onto the µ-slides in a concentration of 3x10⁵ cells/mL in plain RPMI 1640 medium (Sigma-Aldrich). Cells were stimulated with 100 nM PMA or 4 µM calcium ionophore. At each time point (1-6 h, every hour) cells were fixed with 4 % formaldehyde for 30 min, and afterwards permeabilized with 0.5 % Triton-X-100 for 5 min. Wells were blocked with 5 % BSA (AppliChem, Darmstadt, Germany) for 1 h and stained overnight with anti-myeloperoxidase antibody in 1:200 dilution in PBS (sc-52707, Santa Cruz Biotechnology, Heidelberg, Germany). Slides were washed 3 times with PBS for 10 min. Staining was completed by a 2 h incubation with Alexa Fluor 488 coupled secondary anti-mouse antibody (1:1000 in PBS, Invitrogen, Thermo Fisher Scientific) and Hoechst 33342 nuclear counterstaining (2 ng/µL in PBS, Thermo Fisher Scientific). After three times washing for 10 min with PBS five images per condition were taken in 10-fold original magnification.