MPs internalisation by HUVECs was examined by confocal microscopy analysis. MPs were labeled with 2 µM PKH26 red fluorescent cell linker kits (Sigma-Aldrich) for 5 min at RT. HUVECs were treated with labeled MPs at concentration 5 × 106 particles/ml for 2 h at 37 °C, 5% CO2. Then non-adherent MPs were removed by gently washing 3 times with PBS. HUVECs were fixed with 4% formaldehyde for 30 min and stained with F-actin staining kit-green fluorescence (Abcam, Cambridge, UK) for 60 min at RT. Slides were washed and mounted using mounting buffer containing DAPI (X-ZELL, Sunnyvale, CA). Serial Z-stacked confocal imaging was performed with an A1R confocal microscope (Nikon, Tokyo, Japan), and image analysis was performed with NIS Element Viewer 4.2 software (Nikon). Single representative images of the central layers are shown.
Nis element viewer 4
Manufactured by Nikon
Sourced in Japan
NIS Element Viewer 4.2 is a software application developed by Nikon. The core function of this software is to allow users to view and analyze digital images captured using Nikon's imaging equipment.
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Lab products found in correlation
2 protocols using nis element viewer 4
1
Internalization of MPs by HUVECs
2
Isolation and Characterization of Platelet-Derived Microvesicles
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Peripheral blood MPs were prepared as described in the previous study. 14 Briefly, 10 ml of fresh whole blood samples collected in 3.2% trisodium citrate anticoagulant were centrifuged at 1,500 × g for 15 min at 25°C to collect platelet-poor plasma and then re-centrifuged at 14,000 × g for 2 min at 4°C to obtain platelet-free plasma (PFP). One hundred microliter of MP pellets were collected after centrifugation of PFP at 14,000 × g at 4°C for another 45 min, washed once with 2 ml of Dulbecco's phosphate-buffered saline (DPBS), and resuspended with 200 μl DPBS. microscope (Nikon, Tokyo, Japan), and image analysis was performed with NIS Element Viewer 4.2 software (Nikon).
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