D6046

CMT 167 cells (kind gift from Prof. Paola Allavena, Humanitas Research Hospital) were cultured in Gibco Dulbecco's Modified Eagle Medium (DMEM), high glucose (Sigma‐Aldrich, #D5796), with 10% Fetal Bovine Serum (FBS, Sigma‐Aldrich #F7524), 2 mM L‐glutamine and 100 U/mL penicillin–streptomycin (Pen/strep). 4T1 cells (CRL‐2539, American Type Culture Collection (ATCC)) were grown in RPMI 1640 Medium with 10% FBS and 100 U/mL Pen/strep. KPCY cells (2838c3, Kerafast) were cultured in DMEM (Sigma‐Aldrich #D5796) with 10% FBS and 100 U/mL Pen/strep. MC38 cells (ENH204, Kerafast) were cultured in low glucose DMEM (Sigma‐Aldrich #D6046) with 10% FBS, 2 mM L‐ glutamine, 1* non‐essential amino acids, 1 mM sodium pyruvate, 10 mM HEPES (4‐(2‐hydroxyethyl)‐1‐piperazineethanesulfonic acid), and 100 U/mL Pen/strep. All chemicals were from Sigma‐Aldrich unless specified. Cells were thawed and maintained in exponential growth for 2–3 weeks at 37°C and 5% CO₂ before implantation.

Human embryonic kidney cells (HEK 293T/17, ATCC-LGC Standards, Manassas, Virginia, USA) were cultured in Dulbecco’s Modified Eagle Medium (DMEM) with low glucose (D6046, Sigma-Aldrich, Hamburg, Germany) supplemented with 10% fetal calf serum (F9665, Sigma-Aldrich, Hamburg, Germany) and 1% penicillin/streptomycin (P4333, Sigma-Aldrich, Hamburg, Germany). HEK cells offer the advantage of being easy to transfect even with large constructs and thus represent a widely used cell culture model for overexpression experiments. We use HEK cells to overexpress the 2521 amino acid protein Piezo1 (Uniprot entry Q92508, Section 2.1.2) and to test the effects of various compounds on Piezo1-induced cell stiffening (Section 2.1.3).
A human atrial fibroblast line (HAF, [36 (link)]) was cultured in DMEM supplemented with 2 mM L-alanyl-L-glutamine (GlutaMAX, 31966021, LifeTechnologies, Darmstadt, Germany), 10% fetal calf serum and 1% penicillin/streptomycin. At ≈90% confluence, cells were detached using Trypsin-ethylene-diamine-tetraacetic acid (59418C, Sigma-Aldrich, Hamburg, Germany) and seeded in fresh polystyrene flasks (Z707538, TPP, Trasadingen, Switzerland) for maintenance, or on various substrates for experiments (see below).

We cultured HeLa cells in Dulbecco’s modified Eagle medium (DMEM; D6046, Sigma-Aldrich) supplemented with 10% fetal bovine serum (FBS; Biowest) at 37 °C in a 5% CO₂ incubator. We seeded cells on in-house-made glass bottom dishes and grew up to 60–70% of cell confluence before performing transfection. For transfection, we mixed 2.0 µg of plasmids and 5.0 µg of polyethylenimine MAX (24765–1, Polysciences) in 200 µL of Opti-MEM medium (31985-070, Thermo Fisher Scientific), and incubated for 20 min at room temperature. Subsequently, we added the mixture to 1 mL of culture medium containing HeLa cells, and exchanged the culture medium at 6 h after the mixing. We cultured the cells for ~ 48 h and exchanged the medium to DMEM/F12 (11039-021, ThermoFisher Scientific) without phenol red before microscopy observation.
To establish a HeLa cell line stably expressing ELP-TEMP or ELP-REF, 2 days after transfection, we exchanged the culture medium with DMEM supplemented with 10% FBS and 500 µg/mL geneticin (G-418, 10131-035, Gibco). We kept culturing the cells until colonies formed. Finally, we isolated a fluorescent colony and grew up in the same medium to increase the cell number. After establishing the stable cell lines, we grew them with a DMEM medium containing 10% FBS and 200 µg/mL geneticin.

Immortalised murine bone marrow-derived macrophages (iBMDM) were a gift from Prof. Douglas Golenbock (University of Massachusetts, USA). [45 (link)] iBMDM were cultured in Dulbecco’s Modified Eagle’s medium (DMEM) (D6046, Sigma Aldrich, Germany) supplemented with 10% Fetal Calf Serum (FCS) (SH3007003 Cytiva Hyclone, Thermo Fisher Scientific, Basingstoke, Hampshire, UK), 2 mM L-glutamine (59202C, Sigma Aldrich, Germany), 100 U/ml penicillin and 100 μg/ml streptomycin (Penicillin-Streptomycin P4458, Sigma Aldrich, Germany). iBMDMs were seeded in 96 well plates at 1 x 10⁶ cells/ml unless otherwise stated.