Biotek synergy h1 hybrid multi mode reader

TF-1 cells (American Type Culture Collection, VA, USA) were purchased
and cultured with the final concentration of 5 ng/ml recombinant hGMCSF
(G5035–5UG, Sigma-Aldrich, MO). To measure cell proliferation, TF-1 cells
were treated with either A192, rhGMCSF, or hGMCSF-A192 with different
concentrations. The concentrations of hGMCSF-A192 were: 47, 9.3, 1.9, 0.37,
0.075, 0.015, 0.0030, 0.00060, 0.00012, and 2.4×10⁻⁵nM. The concentrations of rhGMCSF were: 34, 6.8, 1.4, 0.27, 0.054, 0.011,
0.0022, 0.00044, 8.7×10⁻⁵, and
1.7×10⁻⁵ nM. The concentrations of A192 were: 41,
8.3, 1.7, 0.33, 0.066, 0.013, 0.0027, 0.00053, 0.00011,
2.1×10⁻⁵ nM. The cells were incubated in a 96-well
plate at 37 °C for 72 hours, and Cell Proliferation Reagent WST-1
(5015944001, Sigma-Aldrich, MO) was added to the cells followed by additional 2
hours of incubation. An absorbance value of each well on the 96-well plate was
measured with Biotek Synergy H1 Hybrid Multi-Mode Reader (Biotek, VT, USA) at
450 nm and 690 nm, and the absorbance at 690 nm was subtracted from the
absorbance at 450 nm to measure the biological activity of proteins at each
concentration. The graph was fit to the following equation: $${OD}_{450}-{OD}_{690}=Baseline+C_{protein}\times \frac{Plateau-Baseline}{{EC}_{50}+C_{protein}}$$