Tryple express without phenol red

Cell lines: Human breast cancer cell line (MCF-7), TNBC (MDA-MB-231), and murine metastatic breast cancer cell line (4T1).
Reagents: Dulbecco’s modified eagle medium (DMEM) powder, fetal bovine serum (FBS), penicillin streptomycin, trypLE Express without phenol red and 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) from Gibco BRL (Carlsbad, CA, USA). DMEM media, calcium chloride dihydrate (CaCl₂·2H₂O), sodium bicarbonate (NaHCO₃), potassium phosphate monobasic (KH₂PO₄), sodium phosphate dibasic heptahydrate (H₁₅Na₂O₁₁P), bovine serum albumin (BSA), skim milk powder, tris (hydroxmethyl)amino-methane, phosphate inhibitor cocktail 2, EDTA-free protease inhibitor tablets, dimethyl sulphoxide (DMSO), and thiazolyl blue tetrazolium bromide (MTT), methanol, and trypan blue (0.4%) from Sigma-Aldrich (St. Louis, MO, USA). Sodium chloride (NaCl) and potassium chloride (KCl) from Fisher Scientific (Loughborough, Leicestershire, UK). Sodium dodecyl sulfate (SDS) and Restore PLUS Western blot stripping buffer from Thermo Fisher Scientific (Rockford, IL, USA). Triton C-100, glycine, blotting-grade blocker, Clarity Western ECL substrate, tween-20, bromophenol blue, quickstart Bradford 1X dye reagent, quickstart bovine serum albumin (BSA) standard set, dithiothreitol (DTT), and mini-protean TGX gels from Bio Rad (Hercules, CA, USA). Validated siRNAs from Qiagen (Valencia, CA, USA).

We used hASCs (R7788115; Thermo Fisher Scientific, Waltham, MA, USA) for cell-loaded micropatches. hASCs were cultured in a 10 cm² culture dish containing Alpha modified Eagle’s medium (Gibco, Grand Island, NY, USA), 10% fetal bovine serum (FBS; Gibco), and 1% penicillin–streptomycin (Gibco) at 37 °C in a humidified atmosphere of 5% CO₂ as manufactures protocol. When cells reached 70–80% confluence, they were trypsinized with TrypLE™ Express without phenol red (Gibco), and the seeding density was 5000 cells/cm². The medium was replaced every 3–4 days with fresh subculture. Cells were used for transplantation at passages 5–6.
Before seeding hASCs on the micropatterned heparin-based hydrogel, PEG-SH in PBS (pH 8.0) was grafted on the remaining area of the electrode without hydrogel pattern to prevent the non-selective cell attachment on the substrate. Then, hASCs (2 × 105 cells) were seeded on the electrode with hydrogel micropattern in a 6-well plate (ultra-low attachment). The seeded cells on the micropatterned hydrogels were cultured for 7 days in DMEM containing 10% FBS and 1% penicillin/streptomycin.