Mouse vegf a duoset elisa kit

In macrophage-tumor cell co-culture experiments, 1×10⁶ macrophages were cultured alone or co-cultured with 1×10⁶ MDA-MB-231 tumor cells for up to 48 hours in α-MEM media with 0.5% FBS and 300 units/mL of CSF-1 (one-tenth of CSF-1 added to complete media). Experiments using immortalized Csf1r^+/+ or Csf1r^−/− BMMs used α-MEM media with 0.5% FBS and 2 ng/mL GM-CSF. In mono-culture macrophage ELISA experiments where tumor cell conditioned media was added, conditioned media was collected from tumor cells incubated in α-MEM with 0.5% FBS and 300 units/mL of CSF-1 for 24 hours. In tumor cell mono-culture ELISA experiments, 1×10⁶ tumor cells were seeded and incubated for 24 hours in serum-free DMEM medium.
In all ELISA experiments, blocking antibodies, isotype controls, inhibitors and controls were added at indicated concentrations in “Inhibitors” section, at time zero. Media conditioned by the cultured cells were collected at the indicated time points and stored at −80⁰C until use. ELISAs were performed per the manufacturer’s recommendations using the Mouse VEGF-A DuoSet ELISA kit (cat# DY493, R&D Systems) or the Human M-CSF DuoSet ELISA kit (cat# DY216, R&D Systems). The concentration of protein secreted was interpolated from the standard curve measurements.