Human gm csf duoset

The following ELISA kits were purchased from R&D Systems and used according to the manufacturer’s protocol (human IL-6 DuoSet, catalog no. DY206; human IL-8/CXCL8 DuoSet, catalog no. DY208; human calprotectin/S100A8 DuoSet, catalog no. DY4570; human MPO DuoSet, catalog no. DY3174, human GM-CSF DuoSet, catalog no. DY215; human G-CSF DuoSet ELISA, catalog no. DY214). Serum samples were diluted (1:2 for IL-6, IL-8, GM-CSF, and G-CSF or 1:1000 for MPO and calprotectin) and the concentrations of the respective analytes were determined using the standards supplied by the manufacturer. The absolute concentrations in serum were established by multiplying the determined values by the respective dilution factors.

For the estimation of basal GM-CSF levels, cells were plated and grown in 0.5% FCS supplemented starvation medium for 24 h. For CM-treatments, cells were plated and stimulated with CM or with starvation media as control for 4 h, then washed and starved for 24 h. For cytokine treatments, cells were stimulated with recombinant cytokines for 24 h. Alternatively, cells were co-stimulated with CM and cytokines for 4 h, then starved for 24 h. Cytokines used were—recombinant human interleukin-1α (hIL-1α, 1–50 ng/mL) and recombinant human tumor necrosis factor-α (rhTNF-α, 1−50 ng/mL) (PeproTech, Rocky Hill, NJ, USA).
The supernatants were then collected, centrifuged, filtered and 15-fold (melanoma supernatants) or 30-fold (brain cell supernatants) concentrated at 4000× g using Amicon® Ultra-15 centrifugal filter units (Merck Millipore, Burlington, MA, USA) for 1 h. The fraction (MW > 3 kDa) was used to determine the extracellular levels of GM-CSF by ELISA according to manufacture instructions using the human GM-CSF DuoSet (R&D systems, Minneapolis, MN, USA).