Mem non essential amino acids

MCF-7 cells (ATCC) were maintained with
DMEM (Corning) supplemented with 10% v/v HyClone cosmic calf serum
(VWR Life Sciences Seradigm), 1% MEM essential amino acids (Quality
Biological Inc.), 1% MEM nonessential amino acids (Quality Biological
Inc.), 1 mM sodium pyruvate (Thermo Fisher Scientific), and 48 ng
insulin/mL media (Insulin, human recombinant dry powder, Sigma-Aldrich).
Cells were maintained in T-75 flasks in a humidified incubator at
37 °C and 5% v/v CO₂. Cells were subcultured when
they reached ∼80% confluency by first being washed with 1X
phosphate-buffered saline (PBS: 137 mM NaCl, 10 mM Na₂HPO₄, 27 mM KCl, and 1.75 mM KH₂PO₄ at pH
7.4) containing 3.7 mM EDTA (Corning) for 2 min and followed by 7
min incubation at 37 °C before being reseeded into a new T-75
flask.

MCF-7 human breast cancer cells (ER⁺/PR⁺) were acquired from American Type Culture Collection (Manassas, Va.). Cells were maintained with Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% v/v HyClone Cosmic Calf Serum (Cytiva SH30087.03), 1% MEM Essential Amino Acids (Quality Biological Inc.), 1% MEM Non-Essential Amino Acids (Quality Biological Inc.), and 0.048 μg/ml Insulin (Insulin, Human Recombinant dry powder, Sigma Aldrich). Cells were maintained in either T-75 or T-182.5 (VWR #10062) flasks in a humidified incubator at 37°C and 5% v/v CO2. The cells were subcultured when 80–90% confluent by first washing the cells with 1X phosphate-buffered saline (PBS: 137 mM NaCl, 10 mM Na2HPO4, 27 mM KCl, and 1.75 mM KH2PO4 at pH 7.4) and then detaching the cells with 3 mL of 3.7 mM UltraPure^™ EDTA solution, pH 8.0 (Thermofisher #15575020) diluted in 1X PBS before re-seeding into a new cell culture flask. Cells used for both on-chip and off-chip experimentation were at 80–90% confluence at time of experiment. Prior to exposure to FSS, cells were first washed with 1X PBS, and then detached with 2 mL of Accutase (Invitrogen) to prevent clumping of cells in the syringes and devices.