Igfbp3 duo set

BJ fibroblasts were treated with vehicle (water—8 times in 24 h; 1 in 1,000), doxorubicin (250 nM—1 time 24 h), or abemaciclib (1 μM—8 times in 24 h). After the treatments, cells were cultured with drug‐free normal medium for 8 days. Thereafter, cells from different groups were trypsinized and counted, and 10⁶ cells from each group were replated and incubated with serum‐free medium for 24 h. Then, the CM was collected and centrifuged at 300 g/5 min to remove any floating cell or debris. The CM was kept in −80°C after harvesting and thawed on ice for further analyses or experiments. Human Cytokine Antibody Array (Abcam, ab133997) was used following the manufacturer’s instructions. After the images were taken, density of positive controls from 3 membranes was equalized using Adobe Photoshop, and individual factors from different membranes were compared. High contrast was adjusted to highlight the most interesting SASP factors and to visualize the lowly expressed factors. For ELISA, human CXCL1/GROα duo‐set or CCL5 duo‐set or IGFBP3 duo‐set (R&D Systems) was used to detect the concentrations of CXCL1 or CCL5 or IGFBP3 in the CM following the manufacturer’s instructions.

Paired plasma samples before the start of therapy (pretreatment) and after ± 4 weeks (median 29 days) of treatment (on‐treatment) were obtained from 9 metastatic breast cancer patients who received palbociclib treatment and 10 metastatic breast cancer patients who received paclitaxel treatment. Written informed consent was obtained from all patients. Mean age was 55.9 years (SD 10.9) for the palbociclib group and 60.1 years (SD 7.4) for the paclitaxel group. The samples were collected in cell stabilizing blood collection tubes (CellSave or Streck Cell‐Free DNA BCT tubes). After collection, the blood was centrifuged twice (1700 g for 10 min and 12,000 g for 10 min). Afterward, the plasma was pooled and directly stored in 2‐ml tubes in −80°C until analyzed. For ELISA, human CXCL1/GROα duo‐set or CCL2 duo‐set or CCL5 duo‐set or MMP1 duo‐set or IGFBP3 duo‐set (R&D Systems) was used to detect the concentrations of CXCL1 or CCL2 or CCL5 or MMP1 or IGFBP3 in the plasma following manufacturer’s instructions.