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. 2016 Aug 23;7(38):62439–62459. doi: 10.18632/oncotarget.11516

Figure 1. Purification of CXCL12 from stromal cell conditioned medium.

Figure 1

MS-5 cells were stimulated with IFN-γ and the double-stranded RNA poly I:C in the presence of neutrophils and PBMCs. 72h after stimulation, conditioned medium was collected and purified using two consecutive chromatographic steps. Panel A shows the results from the heparin-Sepharose affinity chromatography. Proteins were eluted using a two-step sodium chloride gradient (dashed line). Total protein content was determined for each fraction using a BCA assay (open triangles). Total CXCL12 concentrations were determined for each fraction by ELISA (open crosses). Panel B shows the purification of the selected heparin-Sepharose fractions (i.e. fraction 29 to 32) by C8 RP-HPLC. Proteins were eluted using a gradient of acetonitrile in 0.1% (v/v) TFA (dashed line) and detected by their UV absorption (λ = 214nm, full line). CXCL12 concentrations were determined by ELISA (open crosses). mAU = milliabsorption units.