Cells were seeded on culture dishes with glass bottom, and images of live cells were taken with a Carl Zeiss LSM 5 LIVE (LSM 510 META and LSM 5 LIVE) DuoScan Laser Scanning microscope immediately after 4 h of MSC-T cell co-culture. 3D reconstruction was done using the Imaris Bitplane software.
Lsm 5 live duoscan laser scanning microscope
Manufactured by Zeiss
Sourced in Germany
The LSM 5 Live DUOScan is a laser scanning microscope developed by Zeiss. It is designed for high-speed, high-resolution imaging of live samples. The microscope features dual-channel scanning capabilities and advanced optics for capturing detailed images of dynamic biological processes.
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4 protocols using lsm 5 live duoscan laser scanning microscope
1
3D Imaging of MSC-T Cell Co-culture
2
Macrophage Uptake of Labeled Extracellular Vesicles
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BMDM and human macrophages were isolated as described above. Cells (3 × 105) were seeded onto 35 mm glass bottom dishes (MatTek Corporation) and allowed to attach overnight. Medium was replaced using phenol red-free, EVs-free DMEM medium and 5 µM Cytochalasin D (Santa Cruz Biotechnology). Cells were incubated for 30 min at 37 °C under 5% CO2. PKH26-labeled EVs (25 µg/MatTek) were added after incubation with Cytochalasin D and cells were incubated for 4–5 h at 37 °C under 5% CO2 to allow binding. Control cells did not receive EVs. Cell plasma membrane was labeled with CellMask™ Green plasma membrane stain (1:1000; Invitrogen) at 37 °C for 5 min. The medium was replaced with phenol red-free, EV-free DMEM medium containing 5 µM Cytochalasin D. Z-stacked images were acquired with a LSM 5 Live DUOScan laser scanning microscope (Zeiss). Composites were constructed using ImageJ (NIH) Z projection (maximum intensity).
3
Live Cell Imaging of EV Uptake
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BMDMs were seeded and treated as described above. Cells were labeled with CellMask™ Green plasma membrane stain (Invitrogen). Medium was replaced with phenol red-free, EV-free DMEM medium containing 5 µM Cytochalasin D. Cells were placed in an external unit and kept at 37 °C under 5% CO2. Z-stacked images were acquired every 5 min for 2 h with a LSM 5 Live DUOScan laser scanning microscope (Zeiss). Twenty-five µg of PKH26-labeled EVs were added after time 0 for the positive control. No EVs were added to the negative controls. Z-projections (maximum intensity) of each channel and videos of the Z projections were constructed in ImageJ at 2 frames-per-second (fps). Arrows were added with Windows Video Maker (Windows).
4
Multimarker Cardiac Tissue Analysis
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The hearts were embedded in OCT (Sakura, Japan) and cut into 8-μm sections. Then, the sections were fixed using cold acetone at -20°C and placed in a fume hood to ventilate for 20 minutes. The slides were gently rinsed twice with PBS, permeabilized with 0.1% Triton X-100 for 10 minutes, rinsed again twice with PBS and blocked using 2% bovine serum albumin (BSA) for 30 minutes. The sections were then incubated at 4°C overnight with the following antibodies diluted in the blocking buffer: c-kit (1/200), Ki67 (1/400), p-Met (1/200), cTnI (1/200), CD45 (1/100), Gata6 (1/200), Ets-1 (1/200) and Nkx2.5 (1/200). Then, the sections were incubated for 1 hour in dark with the secondary antibodies diluted in the same blocking buffer (green, 1/200 dilution of fluorescein; red, 1/400 dilution of CY3 or rhodamine-labelled secondary antibodies), followed by three washes with PBS. The sections were then mounted with VECTASHIELD mounting medium containing diamidino-2-phenylindole (DAPI), (Vector Laboratories, Burlingame, CA, USA), and images were acquired using an LSM 5 Live DuoScan Laser Scanning Microscope (Zeiss, Oberkochen, Germany).
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