Singlequots supplement pack

Chemotaxis assay was performed using Boyden chambers (BD Biosciences) as previously described (Inaguma et al, 2011 (link)). In brief, cells pre‐treated with either AMD3100 (SIGMA) or vehicle were resuspended in DMEM containing 0.5% BSA and seeded in the top chamber. Medium containing 100 ng/ml CXCL12 was added to the bottom chamber as an attractant. Twenty‐four hours (MCF‐7) or 18 h (4T1‐Luc) after application, migrated cells were stained and counted. For mammosphere assay, 3,000 cells were resuspended in 400 ml of MEBM basal medium (LONZA) supplemented with bovine pituitary extract, human epidermal growth factor, insulin, hydrocortisone, gentamicin, and amphotericin (SingleQuots supplement pack, LONZA) and cultured in 24‐well ultra‐low attachment plate (Costar). To estimate the effect of CXCL12/CXCR4 pathway, 100 ng/ml of CXCL12 and 12 mM of AMD3100 were applied. After 6 days, the number of mammosphere with a diameter more than 100 mm was counted.

Human embryonic kidney cells (HEK293) and human bronchial epithelial cells (BEAS-2B) were purchased from ATCC (Manassas, VA, USA). HEK293 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Invitrogen, Carlsbad, CA, USA), supplemented with 10% fetal bovine serum (Gibco, ThermoFisher, Waltham, MA, USA), 10 U penicillin/mL, and 10 g streptomycin/mL (ThermoFisher Scientific, Waltham, MA, USA). Cells were maintained in 10 cm tissue culture plates at 37 °C under 5% CO₂. BEAS-2B cells were cultured in either DMEM (Invitrogen, Carlsbad, CA, USA), supplemented with 10% fetal bovine serum (Gibco, ThermoFisher) for the short term treatments, or 1× Bronchial Epithelial Cell Growth Medium (BEGM) (Lonza, Basel, Switzerland) SingleQuots Supplement Pack (Lonza, Basel, Switzerland) containing 2 mL BPE, 0.5 mL Insulin, 0.50 mL Hydrocortisone, 0.5 mL GA-1000, 0.5 mL Retinoic Acid, 0.5 mL Transferrin, 0.5mL Triiodothyronine, 0.5 mL Epinephrine, and 0.5 mL hEGF for the long term treatments. Cells were maintained in 6-well tissue culture plates at 37 °C under 5% CO₂. The BEAS-2B cells cultured for 9 weeks in BEGM were maintained in 6-well tissue culture plates coated with a 2:1 mixture of 0.1% gelatin (s006100, Gibco, ThermoFisher) and 0.01 mg/mL (Gibco, ThermoFisher).