Confocal microscopy was used to visualize the cells. The cells seeded on scaffolds were fixed with methanol (−20 °C), rinsed with PBS and stained. 3,3’-Dihexyloxacarbocyanine iodide (DiOC6(3)) was used to visualize the cellular membranes (Life Technologies, Carlsbad, CA, USA; 1 μg/mL in PBS, 30 min) and propidium iodide (Sigma-Aldrich, MO, USA; 5 μL/mL, 10 min) to visualize the cell nuclei. Between the incubations, the samples were rinsed with PBS. A Zeiss LSM 510 DUO confocal microscope was used for imaging (λex maximum = 488 nm, λem maximum = 501 nm for DiOC6(3); λex maximum = 536 nm, λem maximum = 617 nm for propidium iodide).
Lsm 510 duo confocal microscope
Manufactured by Zeiss
Sourced in Germany
The LSM 510 DUO is a confocal microscope designed for high-resolution imaging. It features dual-channel detection and a range of imaging modes, including fluorescence and transmitted light. The microscope is equipped with a range of laser sources and can be used for a variety of applications in the life sciences and materials research.
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6 protocols using lsm 510 duo confocal microscope
1
Confocal Microscopy of Cellular Scaffolds
2
Cell Membrane and Nucleus Visualization
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On days 1, 7, and 14, the cells seeded on the scaffolds were fixed with methanol (−20°C), washed with PBS, and stained with DiOC6 (1 μg/mL in PBS; 30 min at RT; green color, wavelength maxima λex=488 nm, λem=501 nm; Thermo Fisher Scientific) to visualize the cell membranes and with propidium iodide (5 μL/mL in PBS; 10 min at RT; red color, wavelength maxima λex=536 nm, λem=617 nm) to visualize the cell nuclei. The samples were scanned using LSM 510 DUO confocal microscope (Zeiss, Oberkochen, Germany).
3
Immunostaining of Cells on Coated Coverslips
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Cells plated onto laminin- and poly-l -ornithine-coated coverslips were fixed and immunostained according to the protocol described previously [16 (link)]. The primary antibodies used in the study are listed in Table A3 . NPs-iPS blocked with normal goat serum and incubated only with secondary antibodies served as a negative control. Cell nuclei were stained by incubation with DAPI in PBS (5 min) at 24 °C, mounted with Aqua Poly/Mount and imaged using a ZEISS LSM 510 DUO confocal microscope (Zeiss, Oberkochen, Germany).
4
Confocal Microscopy of Fixed Hearts
5
Cell Membrane and Nuclei Visualization
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The cells were fixed with methanol (−20 °C), washed with PBS and stained with DiOC6(3) (Cat. No. 318426, Sigma-Aldrich, St. Louis, MO, USA), 1 μg/mL in PBS; 30 min at RT; green color) to visualize the cell membranes and propidium iodide (Cat. No. P4170, Sigma-Aldrich, St. Louis, MO, USA, 5 μL/mL in PBS; 10 min at RT; red color) to visualize the cell nuclei. The samples were scanned using LSM 510 DUO confocal microscope (Zeiss, Oberkochen, Germany) at λex = 488 nm, λem = 505–555 nm for DiOC6(3), λex = 560 nm, λem > 575 nm for propidium iodide, Objective × 20.
6
Visualizing Cell Viability and Morphology
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The cells were visualized using confocal microscopy. First, the samples were fixed using frozen methanol and stained with DiOC-6 (Life Technologies, USA, CA) to visualize cell biological membranes, and propidium iodide (Sigma-Aldrich, Germany) to visualize cell nuclei. The cell area was determined using Ellipse software (Ellipse, Slovak Republic) from at least 3 independent images. Then, to visualize the viability of the cells, living cells were stained using BCECF-AM (Sigma-Aldrich, Germany) and propidium iodide to visualize dead cells. Viable cells metabolized nonfluorescent BCECF-AM to fluorescent BCECF (green color). propidium iodide does not penetrate into living cells, therefore only dead cell had red nuclei. The cells were counted using ImageJ software (NIH, USA) from at least 3 independent images, the viability of cells is expressed as percentage of viable cells of the total number of cells present on the scaffolds. The stained samples were observed using LSM 510 DUO confocal microscope (Zeiss, Germany). DiOC-6 (green color; maximum excitation wavelength 484 nm, maximum emission wavelength 501 nm), propidium iodide (red ORIGINAL RESEARCH color; maximum excitation wavelength 536 nm, maximum emission wavelength 617 nm), BCECF-AM (green color; maximum excitation wavelength 490 nm, maximum emission wavelength 535 nm).
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