Journal: Methods (San Diego, Calif.)

Article Title: Isolation and culture of adult murine cardiac atrial and ventricular fibroblasts and myofibroblasts.

doi: 10.1016/j.ymeth.2021.04.004

Figure Lengend Snippet: Fig. 3. Phase contrast images of P0 atrial and ventricular fibroblast and myofibroblast monolayer at different time points. Cardiac fibroblasts isolated from mouse and rat heart (ventricles and atria) were seeded onto ‘non-compliant’ uncoated standard polystyrene cell culture dishes with an elastic modulus of ≈107 kPa or ‘compliant’ collagen-coated hydrogel-bound polystyrene plates with a much lower elastic modulus of 8 kPa. (A) Mouse ventricular fibroblasts (8 kPa) and myofi broblasts (107 kPa) imaged at the indicated time after plating. (B) Mouse atrial fibroblasts (8 kPa) and myofibroblasts (107 kPa) imaged at the indicated time after plating. (C) Rat ventricular myofibroblasts (107 kPa) imaged 150 min (0 h) after isolation. (D) Rat atrial myofibroblasts (107 kPa) imaged 150 min (0 h) after isolation. Scale bar = 50 μm.

Article Snippet: Ventricular or atrial cell suspensions are then plated onto two ‘compliant’ 35 mm collagen-coated soft hydrogel-bound polystyrene plates (elastic modulus 8 kPa; Matrigen PS35-COL-8) or ‘non-compliant’ uncoated standard 35 mm culture dishes (elastic modulus ≈107 kPa) to obtain quiescent fibroblasts or activated fibroblasts (myofibroblasts), respectively, and incubated in a humidified incubator at 37 ◦C in a 5% CO2 atmosphere for 150 min (“pre-plating”).

Techniques: Isolation, Cell Culture

Journal: Methods (San Diego, Calif.)

Article Title: Isolation and culture of adult murine cardiac atrial and ventricular fibroblasts and myofibroblasts.

doi: 10.1016/j.ymeth.2021.04.004

Figure Lengend Snippet: Fig. 6. The abundance and cytoskeletal incorporation of smooth muscle α-actin immunoreactivity are attenuated when cardiac fibroblasts are cultured on a compliant substrate. (A) Cardiac fibroblasts isolated from mouse ventricles were seeded onto ‘non-compliant’ uncoated standard cell culture dishes (elastic modulus 107 kPa) or ‘compliant’ collagen-coated hydrogel-bound polystyrene plates (elastic modulus 8 kPa). Lysates (20 μg/lane) from passage 2 cells were used for immunoblot assays. A representative immunoblot showing the abundance of smooth muscle α-actin (αSMA) immunoreactivity from 1 of 3 independent experiments is shown on the left. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as a loading control. Numbers on the right of the immunoblot indicate the position of molecular mass markers (in kDa). The histogram on the right shows the mean ± SEM of three independent experiments. (B) Cardiac fibroblasts were seeded onto either ‘non-compliant’ uncoated glass coverslips in standard polystyrene culture dishs (elastic modulus 107 kPa) or ‘compliant’ collagen-coated soft hydrogel-bound glass coverslips (Matrigen #SS6-COL-8) inserted into compliant culture dishes from Matrigen (elastic modulus 8 kPa). Passage 2 cells were fixed with 2% paraformaldehyde, permeabilized, and decorated with Alexa Fluor 555-phalloidin (to identify actin filaments) and antibodies to αSMA. The images were acquired using a Zeiss LSM-510 confocal fluorescence microscope. Nuclei were visualized using the DNA-binding fluorescent dye, DAPI, and overlay images of the staining are shown (Merge). Images are representative of three independent biological replicates. Scale bar = 20 μm.

Article Snippet: Ventricular or atrial cell suspensions are then plated onto two ‘compliant’ 35 mm collagen-coated soft hydrogel-bound polystyrene plates (elastic modulus 8 kPa; Matrigen PS35-COL-8) or ‘non-compliant’ uncoated standard 35 mm culture dishes (elastic modulus ≈107 kPa) to obtain quiescent fibroblasts or activated fibroblasts (myofibroblasts), respectively, and incubated in a humidified incubator at 37 ◦C in a 5% CO2 atmosphere for 150 min (“pre-plating”).

Techniques: Cell Culture, Isolation, Western Blot, Control, Fluorescence, Microscopy, Binding Assay, Staining