Cytokine Profiling of HBV Antigen-Stimulated Blood

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).