Fgm 2 fibroblast growth medium 2 bulletkit

Force spectroscopy measurements and elasticity maps from cells were collected by using a commercially available atomic force microscope (model XE-120, Park System). The PDMS samples covered with a monolayer of cells were placed inside the Petri dish lid and filled with cell culture medium (FGM–2 Fibroblast Growth Medium-2 BulletKit, Lonza, catalog number: CC-3132). Samples prepared in this way were mounted on the AFM. The ORC8-10 D AFM probe was used, a commercially available silicon nitride from Bruker. The tip was immersed in medium and approached close to the cell’s surface. Force curves (i.e., vertical deflection vs. scanner position) were collected within a grid of 5 × 5 points from approximately 40 different cells, for each substrate and time point. To estimate the quantity of the relative Young’s modulus, only the approach section of the collected force curve was analyzed. It was recalculated into a force versus indentation curve. After that, the Hertz contact model was fitted, with a paraboloid approximation of the shape of the probing tip [97 (link)].

The following reagents were obtained: FGM-2 Fibroblast Growth Medium-2 Bullet Kit (Lonza, Basel, Switzerland); 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT, Sigma-Aldrich, St Louis, MO, USA); PBS (Lonza, Basel, Switzerland); dimethyl sulfoxide (Sigma-Aldrich, St Louis, MO, USA); trypan blue (Invitrogen, Carlsbad, CA, USA); TRIzol reagent (Invitrogen, Carlsbad, CA, USA); SensiFAST SYBR No-ROX One-Step (Bioline, London, UK); and oligonucleotide specific primers (Sigma-Aldrich, St Louis, MO, USA).

Force spectroscopy measurements and the collection of elasticity maps from cells were carried using an atomic force microscope (AFM, model XE-120, Park System, Suwon, Korea) with commercially available silicon nitride AFM probes (ORC8-10 D, Bruker, Bremen, Germany). The samples covered with a monolayer of cells were placed inside a Petri dish lid, filled with cell culture medium (FGM–2 Fibroblast Growth Medium-2 BulletKit, Lonza, Basel, Switzerland, catalog number: CC-3132), and mounted on the AFM. The tip then approached close to the cell surface and force curves were collected within a grid of 5 × 5 points from about 40 different cells, for each substrate and time point. To estimate the quantity of the relative Young’s modulus, only an approach part of the collected force curve was analyzed using the Hertz contact model, with a paraboloid approximation of the probing tip shape [101 (link)].

The human alveolar epithelial cell line A549 and diseased human lung fibroblasts from patients with idiopathic pulmonary fibrosis (DHLF-IPF cells) were obtained from the Korean Cell Line Bank (Seoul, South Korea) and Lonza (Basel, Switzerland), respectively. A549 cells were cultured in RPMI-1640 medium (Gibco Cell Culture, Carlsbad, CA, USA) containing 10% fetal bovine serum (FBS; Gibco) and 1% penicillin–streptomycin (Gibco), and DHLF-IPF cells were grown using an FGM-2 Fibroblast Growth Medium-2 BulletKit (Lonza). For hypoxic incubation, cells were incubated in a hypoxic incubator (New Brunswick Scientific, Edison, NJ, USA) with a humidified environment consisting of 1% O₂, 5% CO₂, and 94% N₂.

A chiPSC1.5 iPSC cell line was derived from normal female adult human dermal fibroblast (Lonza CC-2511). Briefly, an early passage (P2) was plated at 3500-cells/cm² in FGM-2 fibroblast growth medium-2 BulletKit (Lonza, CC-3132). Reprograming of human fibroblasts was achieved by using ReproRNA-OKSGM a single-stranded RNA replicon vector that contains the five reprograming factors: OCT4, KLF-4, SOX2, GLIS1 and c-MYC (Stem Cell Technologies (STC) 05930). The manufacturer’s protocol was followed for reprograming the human fibroblasts. Peripheral blood mononuclear cells (PBMCs) were isolated from peripheral blood a female patient (Støy et al., 2008) carrying the KCNJ11 mutation V59M. PBMCs were reprogramed by the Cincinnati Children’s Hospital Pluripotent stem cell facility. Following adaptation to mTeSR1 and matrigel conditions, cell lines were cryopreserved and tested for mycoplasma (PromoKine PK-CA20-700–20). Standard metaphase spreads and G-banded karyotypes were determined by Wi-Cell Laboratories (Madison, WI).