Penicillin

The human breast cancer cell lines MCF-7 and MDA-MB-231 were obtained from the Korea Cell Line Bank (KCLB, Seoul, Korea). Both cell lines were grown in Dulbecco’s Modified Essential Medium (DMEM; HyClone, Logan, UT, USA) supplemented with 10% fetal bovine serum (FBS; HyClone), 100 U/mL of penicillin, and 100 μg/mL of streptomycin (HyClone), and were maintained at 37 °C in a humidified incubator containing 5% CO₂. We used breast cancer cell lines up to 15 passages after purchasing cell lines from KCLB and the cell lines were authenticated by the KCLB and showed >90% similarity in a short tandem repeats (STR) DNA fingerprint profile when compared with the American Type Culture Collection (ATCC) and Korean Cell Line Bank (KCLB) databases. Cells were plated at a density of 1 × 10⁶ cells in 10-cm culture dishes. To establish primary mammospheres, single-cell suspensions of MCF-7 and MDA-MB-231 cells were seeded at a density of (3.5∼4) × 10⁴ and (0.5∼1) × 10⁴ cells/well, respectively, in ultralow attachment six-well plates containing 2 mL of complete MammoCult^TM medium (StemCell Technologies; Vancouver, BC, Canada), which was supplemented with 4 μg/mL of heparin, 0.48 μg/mL of hydrocortisone, 100 U/mL of penicillin, and 100 μg/mL of streptomycin. The cells were incubated for seven days in a 5% CO₂ incubator at 37 °C.

Human breast cancer cells, MCF-7, were obtained from the American Type Culture Collection (ATCC; Manassas, VA, USA). MCF-7 cells were grown in Dulbecco’s Modified Essential Medium (DMEM; Hyclone, Logan, UT, USA) with 10% fetal bovine serum (FBS; Hyclone), 100 U/ml penicillin, and 100 μg/ml streptomycin (Hyclone). Cells were maintained at 37 °C in a humidified incubator with 5% CO₂. To establish primary mammospheres, MCF-7 cells were seeded at a density of 3.5–4 × 10⁴ cells/well in ultra-low attachment 6-well plates containing 2 ml of complete MammoCult™ medium (StemCell Technologies, Vancouver, BC, Canada) which was supplemented with 4 μg/ml heparin, 0.48 μg/ml hydrocortisone, 100 U/ml penicillin, and 100 μg/ml streptomycin. To establish secondary mammosphere, primary mammosphere was collected into individual conical tubes by gentle centrifugation at 1000 rpm for 1 min. After removal of supernatant, cell pellet was trypsinized with 1X EDTA/Trypsin, followed by replating a single cell suspension at a density of 4 × 10⁴ cells/well in ultra-low attachment 6-well plates containing 2 ml of complete MammoCult™ medium [7 (link)]. In 5 days, the number and size of the mammosphere were assessed compared with control. The same procedure was repeated for tertiary mammospheres [7 (link)].

For the MDR assays, daunorubicin (DNR, Sigma-Aldrich, St. Louis, MO) and cyclosporine A (CsA, Sigma-Aldrich) were dissolved in dimethyl sulfoxide (DMSO, Sigma-Aldrich) to make stock solutions of 350 μM and 1 mM, respectively. Working solutions of 35-μM DNR and 5-μM and 10-μM CsA were prepared by diluting stock solutions in Hanks’ Balanced Salt Solution (HBSS, Invitrogen Corp., Grand Island, NY). Working solutions of 1-μM Oregon Green-labeled paclitaxel (OG-PTX) and 10-μM CsA were prepared by diluting stock solutions in a similar fashion. Post-assay cell viability was tested using 0.4% trypan blue solution (ThermoFisher Scientific, Waltham, MA).
The MDA-MB-231 breast cancer cell lines were obtained from cryopreserved storage. The cells were maintained in Hyclone Dulbecco’s Modified Eagle Medium (DMEM)/High Glucose (GE Life Science, Marlborough, MA) with 10% fetal bovine serum (FBS, Life Technologies, Grand Island, NY) and 1% penicillin (Stemcell Technologies, Vancouver, BC) at 5% CO₂ and 37 °C. The doubling time for the cell lines was ~38 h; hence, growth medium was changed three times a week and cells were passaged every 72 h.