Z gel

Mouse serum was prepared from blood collected by cardiac puncture, immediately placed into clotting-activator containing microtubes (1.1 mL Z-gel, Sarstedt), allowed to coagulate for 30 min at room temperature and centrifuged (10,000 rpm, 2 min). OVA-specific antibodies were measured in the serum of tumor-bearing mice by ELISA. Ten-fold serial dilutions of serum samples were added to Iimmunoplates coated with ovalbumin (100 μg/mL) and OVA-specific IgG was detected using a mouse-specific anti-IgG antibody. EC₅₀ values for each serum sample were calculated from the titration curves using an algorithm supplied by the ELISA plate reader (SoftMax Pro) and mice whose titer could not be calculated or was above 0.15 were excluded from the analysis.

The cross-sectional KarMeN study was conducted between March 2012 and July 2013 at the Division of Human Studies of the Max Rubner-Institut in Karlsruhe, Germany. Details on inclusion and exclusion criteria, as well as a comprehensive description of the study design and examination procedures, have already been published [26 (link)]. Briefly, 301 healthy, non-smoking individuals (172 men, 129 women) between 18 and 80 years of age were included. All subjects visited the study center three times and were thoroughly characterized by anthropometric, clinical, and functional examinations. Moreover, data on PA, diet and the menopausal status of female subjects were collected. Since the menstrual cycle is known to affect metabolite profiles [66 (link)], all premenopausal women were scheduled for examinations within their luteal phase. On the morning of the second study day, fasting plasma samples were collected using 9 mL EDTA plasma tubes (S-Monovette, Sarstedt, Nümbrecht, Germany). The plasma samples were immediately centrifuged at 1850× g at 4 °C, aliquoted into small portions, and cryopreserved at −196 °C until metabolomics analyses. Serum samples (S-Monovette Z-gel, Sarstedt, Nümbrecht, Germany) were collected for standard clinical biochemistry analyses.

Serum samples described as normal mouse serum (NMS) and activated zymosan were purchased from Complement Technologies. C57BL/6J blood was isolated by cardiac puncture, and the samples were processed with Z-gel (Sarstedt) to recover serum. All serum was snap-frozen and stored at −80°C when appropriate. Zymosan-activated C57BL/6J mouse serum (zB6MS) was generated by addition of 1:20 activated zymosan (Comptech), followed by incubation at 37°C for 1 h.

Blood was collected by retro-orbital bleeding through a sodium heparin-coated micro hematocrit capillary (Hirschmann Laborgeräte) into a micro tube 1.1ml Z-Gel (Sarstedt). After standing at room temperature for 30 min, the blood was centrifuged at 10 000 × g for 5 min, and serum was transferred to new tubes and stored at −20°C until analysis.

Sample processing began with a 15-mL sample of whole-blood obtained by venipuncture collected in two EDTA tubes (S-Monovette 4.9 ml K3E, Sarstedt, Nümbrecht, Germany) and another tube for serum obtention (S-Monovette 4.9 ml Z-Gel, Sarstedt, Nümbrecht, Germany). Subsequently, the samples were centrifuged for 5 min at 4,500 rpm to separate the peripheral blood mononuclear cells (PBMCs) and the serum, and the serum was stored in cryopreservation tubes at −80°C until use.