Ultra low attachment petri dishes

The generation of in vitro bone marrow-derived MDSCs (BM-MDSCs) was adapted from Marigo et al [29 (link)]. BMDCs were flushed from the femur and tibia of mice and cultured for 5 days in ultra-low attachment petri dishes (Corning) with complete RPMI medium supplemented with β₂-mercaptoethanol and 40 ng/ml GM-CSF and IL-6 (Peprotech) on day 1, 3 and 5. On day 6, BMDCs positive for CD11b, Ly6C and Ly6G (classified as BM-MDSCs) were obtained by Fluorescence Activated Cell Sorting (FACS) as described below and used for subsequent experiments.

The OVCAR-3 cell line was obtained from the Developmental Therapeutic Program (DTP) of the National Cancer Institute (NCI). The OVCAR-3 ovarian cancer cells (OCCs) were cultured in R10 medium: RPMI-1640 (Cellgro, Mediatech Inc., Manassas, VA) supplemented with 10% fetal bovine serum (FBS, Invitrogen, Grand Island, NY) and 1% antibiotic-antimycotic solution (Cellgro, Mediatech Inc., Manassas, VA). Authenticity of the OVCAR-3 cell line was confirmed using short tandem repeat profiling performed by IDEXX RADIL (Columbia, MO) in October 2013. Cells were grown until confluence and subcultured at a ratio of 1:4.
Ovarian cancer stem cells (OCSCs) previously derived from a side population of OVCAR-3 were cultured as previously described [9 (link)]. Briefly, the OCSCs were cultured in ultra-low attachment petri dishes (Corning Incorporated, Corning, NY) in stem cell medium: DMEM/F12 (1:1) (Cellgro, Mediatech Inc., Manassas, VA) supplemented with 0.4% bovine serum albumin (BSA, Sigma-Aldrich, St. Louis, MO), 20 ng/mL epidermal growth factor (EGF, Invitrogen, Grand Island, NY), 10 ng/mL basic fibroblast growth factor (bFGF, Sigma-Aldrich, St. Louis, MO), 5 μg/mL insulin (Sigma-Aldrich, St. Louis, MO), and 1% antibiotic-antimycotic solution (Cellgro, Mediatech Inc., Manassas, VA). The spheroids were dissociated and reseeded at a density of 10⁵ cells/mL each week.