Calcein am

Propidium iodide (PI, Dojindo, Japan), calcein-acetoxymethyl (calcein-AM, Dojindo) and Hoechst 33342 (Dojindo) were incubated with the cells at 37 °C and 5% CO₂ for 15 min at a final concentration of 1, 1.5, and 3.6 μM, respectively, and then were washed once for Inverted Fluorescence Microscope analysis or photographed by a high-throughput cell imaging system at the following fluorescent wavelengths: PI, λex = 530 nm and λem = 580 nm; calcein-AM, λex = 490 nm and λem = 515 nm; and Hoechst33342, λex = 350 nm and λem = 460 nm. Trypan Blue (Sigma, USA) staining was performed at a final concentration of 0.01% in cell medium and then observed under optical light using an inverted microscope.

PC12 cells were seeded on the 24-well plate at 1 × 10⁴ cells. One to two days after seeding, the cells were treated with Pd(bpy) or Pd(sulfo-bpy) for 3-4 days. To visualize the live cells, the cells were loaded with 2 µM calcein-AM (dojindo) for 15 min in HBS, and then washed with HBS. The fluorescent images of the cells were obtained with fluorescent microscopy (Axio Observer7, Carl Zeiss) equipped with CMOS camera (Axiocam712mono, Carl Zeiss), and the cell numbers were automatically counted with ZEN blue software (Carl Zeiss).

AF488 N-hydroxysuccinimide (NHS) ester and AF647 NHS ester were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Rhodamine 6G (Rh6G) was obtained from Sigma-Aldrich (St. Louis, MO, USA). Calcein AM was obtained from Dojindo (Osaka, Japan). SiR-Actin and the calcium channel inhibitor verapamil were obtained from Cytoskeleton (Denver, CO). The TGF-β receptor I inhibitor LY364947, PI, DX, and phenylmethylsulfonyl fluoride (PMSF) were obtained from Fujifilm (Tokyo, Japan). Rabbit polyclonal anti-fibronectin 1 antibody was purchased from Abcam (ab2413) (Cambridge, UK). pAcGFP1-Mem was purchased from Clontech (Takara Bio, Shiga, Japan). Smear Gell (product number: SG-01) for spheroid immunofluorescence was obtained from GenoStaff (Tokyo, Japan). The SlowFade mountant as a microscope was obtained from Thermo Fisher Scientific. The protease inhibitor cocktail (P8340) was purchased from Sigma-Aldrich.

Cell wound scratch assay was performed as manufacturer’s instruction. Firstly, marker pen was used to draw a line at an interval of 1 cm behind the 6-well plate. Then, about 5 × 10⁵ cells were plated into 6-well plate. Then, a scratch wound was generated using a pipette tip and washed three times with PBS to remove the scratched cells. Subsequently, serum-free medium was added and cells were further cultured in a humidified incubator at 37 °C. Cells were stained with Calcein-AM (Dojindo, China) and imaged at 0, 6, 12, 24, and 48 h. Numbers of fluorescent cell corresponding to wound closure rate were calculated by Image-J software (NIH, Bethesda, MD, USA).

Proliferation data were determined using Cell Count Reagent SF (Nacalai Tesque). The data were analyzed using SoftMax® Pro Microplate Data Acquisition and Analysis software (Version 7.0, Molecular Devices, Sunnyvale, CA, USA).
For live and dead staining, HGnFs were stained with calcein-acetoxymethylester (calcein-AM) and propidium iodide (PI) solutions, respectively (Dojindo, Kumamoto, Japan). The stained cells were photographed using a BZ-II all-in-one fluorescence microscope (Keyence Corporation, Osaka, Japan).

NHEKs were seeded confluently in two-well culture inserts (ibidi) on glass-bottom dishes (Matsunami). Culture inserts were removed after overnight incubation, followed by washing with PBS. Cells were then cultured in Humedia-KG2, MCDB153 medium, or low chloride MCDB153 medium. After 12 or 24 h of cultivation, calcein-AM (Dojindo) was added to the culture medium to visualize the cells. ImageJ software was used for data analysis. For time-lapse analysis, cells were cultured in a Stage Top Incubator (TOKAI HIT) on a confocal laser scanning microscope (IX83 Olympus) and images were captured every 10 min.