Lithium heparin monovette

Venous blood from healthy donors was collected in sterile 7.5 ml Lithium Heparin Monovettes (Sarstedt, Germany). 0.5 ml of whole blood were then transferred to sterile, pyrogen-free 2 ml tubes (Sarstedt, Germany) and stimulated with HBV antigens (HBsAg 50 µg/ml). Samples stimulated with 0.9% (w/v) NaCl solution served as negative control whereas SEB (1 µg/ml) and CEFT (50 µg/ml) stimulated samples served as positive controls. Glucose (2 mg/ml final concentration; pre-diluted in sterile 0.9% (w/v) NaCl solution) was added to each tube to further enhance cytokine secretion as described previously [3 (link)]. Following titration, the complement factor C3a was used at a final concentration of 0.1 µg/ml whereas C5a was used at a final concentration of 0.75 µg/ml. All tubes were incubated at 37 °C for 24 h. Upon centrifugation plasma supernatants were aspirated, stabilized with 0.045% (w/v) NaN₃ and stored at − 20 °C until cytokine measurement by ELISA. The amount of biomaterial per donor was not always sufficient to perform all stimulations which is why for some donors conditions had to be left out. This explains variations with regard to the group size (n).

We obtained blood from 26 different NSCLC patients. Blood of lung cancer patients was drawn directly before tumor resection (TP1), around two weeks after tumor resection (TP2) and then around three months (TP3), six months (TP4), nine months (TP5), 12 months (TP6), 15 months (TP7) and 18 months (TP8) after tumor resection. From 3 patients we obtained only blood from 7 time points and from one patient we obtained blood only from 6 time points. In a follow-up of 4 years, 18 patients were free of metastases or recurrences. In addition, we obtained blood from 12 patients from the same clinic that did not suffered from lung cancer but from other non-tumor lung diseases. Blood of all patients was drawn in Lithium-Heparin monovettes (Sarstedt). Plasma was isolated by centrifugation at 3000 rpm for 10 min and stored at −80°C until use. Samples were collected with patient informed consent. The local Ethics Committee approved the study (Ärztekammer des Saarlandes, 01/08). Patient details are provided in Supplemental Table 5.

After overnight fasting, 15 mL of blood was collected in 7.5 mL lithium-heparin monovettes (Sarstedt, Nürnbrecht, Germany) by venipuncture. Isolation of PBMC using Biocoll Separating Solution 1.077 g/mL (Biochrom, Berlin, Germany) was performed as described elsewhere [27 (link)]. Cells were adjusted to a final concentration of 1 × 10⁶ cells/mL in culture medium. The culture medium consisted of RPMI 1640 containing 10% fetal calf serum (FCS, Bio&Sell, Feucht, Germany), 2 mM L-glutamine, 100 U/mL potassium penicillin and 100 U/mL streptomycin sulfate (all Sigma-Aldrich, Steinheim, Germany). Incubation steps were carried out in a humidified 5% CO₂ atmosphere at 37 °C.