Human breast cancer cells (MDA-MB-231, MCF-7, and BT-474) were subjected to treatment with various concentrations of cobalt(II) chloride (CoCl2) (0, 50, 100, 200, and 300 μM) for different time intervals (24 and 48 hours) and the levels of 41 cytokines secreted in conditioned cell culture medium were measured by MILLIPLEX assay.
Cell Lines: MDA-MB-231 (Cat. # HTB-26) and MCF-7 (Cat. # HTB-22) cells were purchased from the American Type Culture Collection (ATCC), and BT-474 were a generous gift from Dr. Dipali Sharma’s laboratory at the Sidney Kimmel Comprehensive Cancer Center at the Johns Hopkins University in Baltimore, Maryland. All cells were grown in Dulbecco's Modified Eagle's Medium (DMEM) (Cat. # 10-013-CF, Corning), supplemented with 10% fetal bovine serum (FBS) (Cat. # 1500-500, VWR International) and antibiotic/antimycotic solution (containing 10,000 IU penicillin, 10,000 μg/mL streptomycin, and 25 μg/mL amphotericin B) (Cat. # 30-004-CI, Corning, Inc.) at 37 °C/5% CO2.
Reagents: Cobalt(II) chloride (CoCl2) (Cat. # 409332, Sigma-Aldrich) anhydrous beads were reconstituted in PBS to a concentration of 250 mM and immediately before treatment, further diluted with serum-free DMEM to desired concentrations (1,000x) used for treatment.
MILLIPLEX assay: Cytokine levels in conditioned cell culture medium were measured by MILLIPLEX assay (Cat. # HCYTMAG-60K-PX41, MilliporeSigma) using MAGPIX Multiplexing System (MilliporeSigma). The kit included the following cytokines: EGF, FGF-2, TGFα, PDGF-AA, PDGF-AB/BB, TNFα, TNFβ, IFN-α2, IFN-γ, CCL2, CCL3, CCL4, CCL5, CCL7, CCL11, CCL22, CXCL1, CXCL10, CX3CL1, IL-1α, IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12p40, IL-12p70, IL-13, IL-15, IL-17A, IL-1RA, G-CSF, GM-CSF, FLT3L, sCD40L, VEGF-A. Data were calculated using xPONENT 4.2 and Milliplex Analyst 5.1 data analysis software [MilliporeSigma].
