Proteome profiler human cytokine and angiogenesis array kits

To establish the composition of CAFs secretome a Proteome Profiler Human Cytokine and Angiogenesis Array Kits (R&D Systems) were utilized. The tests allow for the detection of 36 or 55 different cytokines and chemokines or angiogenesis-related proteins, respectively, thanks to antibodies spotted on the nitrocellulose membrane. The experiment was conducted according to the manufacturer’s protocol. Briefly, conditioned media collected from control cells, fibroblasts treated with CM from WM1341D, and WM9 (each sample containing 25 µg of protein) were mixed with biotinylated detection antibodies. These mixes were then added onto nitrocellulose membranes containing primary antibodies dots and incubated overnight. Next, the membranes were washed, and the signal was detected using streptavidin-HRP. A chemiluminescent signal was measured using ChemiDoc Imaging System (BioRad) and analyzed with ImageLab software (Bio-Rad). The densitometric signal was background corrected and then normalized to a reference spot (mean) for each membrane.

We investigated the composition of the CAA secretome using Proteome Profiler Human Cytokine and Angiogenesis Array Kits (R and D Systems, Minneapolis, Minnesota, USA). The tests allow detection of 36 or 55 different cytokines and chemokines or angiogenesis-related proteins, respectively, thanks to spots of antibodies on nitrocellulose membrane. The experiment was conducted according to the manufacturer’s protocol. Briefly, conditioned media collected from control adipocytes, and adipocytes cocultured with WM1341D, A375, and WM9 cells were mixed with biotinylated detection antibodies. These mixes were then dropped on nitrocellulose membranes containing primary antibody dots and incubated overnight. Next, the membranes were washed, and the signal was detected using streptavidin-HRP. The chemiluminescence signal was measured using a ChemiDoc Imaging System (Bio-Rad, Hercules, California, USA) and analyzed with ImageLab software (Bio-Rad, Hercules, California, USA). Densitometric values were background corrected and then normalized to the mean of reference spots for each membrane.