Heparin coated tube

Blood samples were obtained in 7.5 mL heparin-coated tubes (Sarstedt; Nümbrecht, Germany) and analyzed within 2 h. Cells were tested in whole blood samples. For fluorescence-activated cell sorting (FACS) analysis of the whole blood, an erythrocyte FACS lysing solution obtained from Becton-Dickinson (Heidelberg, Germany) was diluted 1 : 10 in bidistilled water. For cytofluorometry, fluorescent-tagged (fluorescein isothiocyanate (FITC) or phycoerythrin (PE)) antibodies were used. For CD80 and CD86 detection, PMNs in the whole blood were stained with 2 μg anti-CD80-FITC and CD86-PE. Cells were analyzed by the MACS Quant Analyzer (MACS) Miltenyi Biotec and Cell Quest software (Becton-Dickinson, Heidelberg, Germany). All results were expressed as percentage of positive cells in the respective gate.

All mice underwent submandibular blood sampling prior to (D₇), during (D₁₃) and after completion (D₂₀) of their treatments. Blood (200μl) was collected in heparin-coated tubes (Sarstedt, Germany), centrifuged for 10 minutes at 2000g to separate blood cell pellets from plasma. While cell pellets were immediately analyzed, plasma samples were stored at -20°C for further analysis. At day 20 after tumor injection, mice were sacrificed by cervical dislocation and single cell suspensions from lung and spleen were prepared. Lungs were first perfused with 5ml PBS and transferred to 1ml Roswell Park Memorial Institute-1640 medium (RPMI-1640, Sigma-Aldrich) containing 300U/ml collagenase-I (Sigma-Aldrich). Tissues were cut to small pieces using scissors, incubated at 37°C for 45 minutes, and finally mechanically reduced using an 18G syringe until single cell suspensions could be passed through a 40μm strainer. Spleens were transferred to 1ml PBS, stamped with the plunger of a 3cc syringe and passed through a 40μm strainer. Cell pellets from blood, lung and spleen, were resuspended in 1ml red blood cell lysis buffer, incubated for 5 minutes, followed by a centrifugation and wash step with PBS before further analysis.

Blood from volunteers (mainly laboratory personal and students) was drawn into heparin-coated tubes (Sarstedt, Nümbrecht, Germany). Neutrophils were isolated by PolymorphPrep™(Axis-Shield, Oslo, Norway) and residual erythrocytes were removed by hypotonic lysis. The remaining cells were suspended in Hanks balanced salt solution (HBSS), containing 0.5% bovine serum albumin (BSA), at a final concentration of 5 × 10⁶ cells/ml. This procedure yielded 85–95% neutrophils as judged by cytofluorometry using CD66b as marker for neutrophils. Written informed consent was obtained from the volunteers and the ethic committee of the University of Heidelberg approved the procedure.

For measurement of glucagon and insulin levels in vivo, 100μl of blood was collected from the tail vein into heparin-coated tubes (Sarstedt, Beaumont Leys, UK). Plasma was separated by sedimentation at 10,000 g for 20 min (4°C). Plasma glucagon or insulin levels were measured in 50 μL aliquots by radioimmunoassay with competitive ¹²⁵I-labelled glucagon or insulin (Millipore, Watford, UK).

Mice fasted overnight were injected with glucose (3-g glucose/kg bodyweight) and blood from the tail vein was collected into heparin coated tubes (Sarstedt) at 0, 15, and 30 minutes. Plasma was separated by centrifugation at 2000g for 10 minutes, 5 μL of blood plasma were used to measure insulin levels using an ultrasensitive mouse insulin ELISA kit (Crystal Chem).

To quantify circulating insulin and GLP-1(1-37) levels, 100μl of blood was collected from the tail vein into heparin-coated tubes (Sarstedt, Beaumont Leys, UK). Plasma was separated by sedimentation at 10,000 g for 10 min. (4°C). Plasma insulin levels were measured in 5μl aliquots and GLP-1(1-37) levels were measured in 10μl aliquots by ELISA kits from Crystal Chem (USA).