Clot activator

Fifteen milliliters of peripheral blood in EDTA-coated tubes and an additional 10 mL in tubes with a clot activator (Sarstedt, Nümbrecht, Germany) were collected. Peripheral blood mononuclear cells (PBMCs) were isolated with the use of density gradient centrifugation from EDTA-coated tubes. Five milliliters of whole blood diluted with 5 mL of normal saline on 5 mL of Ficoll-Paque™ (Milteny Biotec, Bergisch-Gladbach, Germany) was layered in 15 mm tubes and centrifuged at 400× g for 30 min. With the use of a Pasteur pipette, PBMCs were removed from buffy coats. Trypan blue (0.4% trypan blue solution; Sigma-Aldrich, Hamburg, Germany) was utilized to count PBMCs and assay them for viability. Only PBMCs with a viability ≥95% were deemed eligible. Serum from the tubes with a clot activator was frozen and stored at −80 °C until used.

Blood and stool samples were collected 1 day before vaccination and at the designated time points throughout the study period. Fresh stool pellets were frozen at −80°C until use. Blood was collected from the tail vein using capillary tubes with clot activator (Sarstedt). Sera were obtained by clearing the clotted blood with centrifugation at 10,000 × g for 5 min at room temperature and stored at −20°C. Stool samples were prepared as previously described (40 (link)). Briefly, pellets were thawed on ice, transferred to 15-ml conical tubes containing 3 ml of chilled resuspension solution (0.1 mg/ml soybean trypsin inhibitor and a 3:1 mixture of PBS to 0.1 M EDTA), thoroughly homogenized, and centrifuged at 650 × g for 10 min at room temperature. The supernatant was collected and centrifuged once more at 15,300 × g for 10 min at 4°C. Phenylmethylsulfonyl fluoride (PMSF) was added to the supernatant to a final concentration of 2 mM. Stool samples were kept at −20°C or at −80°C for long-term storage.

Venous blood samples were placed into sterile tubes containing a clot activator (Sarstedt, Nümbrecht, Germany) the biochemical measurements. Plasma was separated by centrifuging at 3000 rpm for 10 min at room temperature 10–15 min after blood collection. Fasting plasma glucose, total cholesterol and high-density lipoprotein cholesterol (HDL-C) and were measured at baseline. Triacylglycerols, TC and HDL-C were measured by an enzymatic colorimetric method using the BM Roche/Hitachi 717 analyzer (kits of Roche). Low-density lipoprotein cholesterol (LDL-C) was calculated at baseline using the Friedewald formula (LDL-C = TC − HDL-C − TAG/5). Plasma TAG concentrations were measured in fasting and postprandial blood samples. According to recommendations of the ISO 1994 and 2012 guidelines [19 ], all measurements were done by the same biochemistry, in the same location, using the same analyzer, the same laboratory tools, and under exactly the same conditions.

The amount of C1q in serum and brain tissue was determined by Mouse C1q ELISA kit (HycultBiotech) according to manufacturer’s instructions. Blood samples were drawn from the saphenous vein of naïve 12 P4htm^−/− (six males and six females) and 12 WT mice (six males and six females) directly to Z-Gel micro tubes containing clot activator (Sarstedt AG & Co) and centrifuged at 10,000g for 5 min to obtain serum. Brain tissue lysates were extracted as described in the protein isolation section from eight P4htm^−/− (seven males and one female) and seven WT mice (four males and three females).

Blood samples were taken collected six times during lactation (the first sampling several hours after parturition) from the jugular vein into sterile 9 mL S-Monovette tubes with clot activator (Sarstedt, Nümbrecht, Germany). All sampling procedures were performed by a veterinarian. After centrifugation, the serum samples were removed and frozen at -20 °C and stored until analysis.