Bone Marrow Cell Activation and Cytokine Measurement

Bone marrow cells were cultured in RPMI (Invitrogen, cat#11965118) with 10% FBS, 2mM L-glutamine, 1mM Sodium pyruvate, 10mM HEPES buffer, 1% Non-essential amino acids, 50μM 2-Mercaptoethanol, 1% Pen/Strep, 20ng/ml GM-CSF (Kingfisher, cat# RP0407M) or 20ng/ml M-CSF (Kingfisher, cat# RP0462M). The medium was changed at day 3 and 6. At day 6, cells (1×10⁶) were transferred to a 24-well plate with fresh medium. Cells were activated at day 7 with 10μg/ml CDA, CDG, 2′3′-cGAMP or 5μg/ml Rp-Rp-ssCDA in culture directly. Mouse IFNβ was measured in culture supernatant after 5hrs by ELISA (PBL Bioscience, cat#42410). Separately, BMDM and BMDC were activated with 5μg/ml HSV DNA (Invivogen, cat# tlrl-hsv60n) and Vaccinia virus DNA (Invivogen, cat# tlrl-vav70n) transfected with lipofectamine®2000(27 (link)) and mouse IFNβ was measured in culture supernatant after 5hrs by ELISA. Alternatively, BMDC were activated with Heat kill streptococcus pneumonia (HKSP) (10⁸c.f.u/ml) (Invivogen, cat# tlrl-hksp), LPS from Salmonella (25ng/ml) (Sigma, cat# L7261), Imiquimod (4ng/ml) (Invivogen, cat# tlrl-imqs) or CpG-ODN2395 (8ng/ml) (Invivogen, cat# tlrl-2395). Mouse TNFα and IFNβ were measured in culture supernatant after 5hrs by ELISA.