MSCs were incubated with or without IL-1β at 50 ng/mL for 48 h. Exosomes were isolated from the culture supernatants by differential centrifugation as previously described, and then characterized using a range of analytical techniques.28 (link) Exosome morphology was examined by transmission electron microscopy (JEM-200CX, Hitachi, Tokyo, Japan), and size distribution was measured using a NanoSight NS300 system (Malvern Panalytical, Malvern, UK) after dilution of 10 μL isolated exosomes to 1 mL with filtered PBS. The identity of exosomes was confirmed by Western blotting (WB) with antibodies against the exosome marker proteins CD63 and TSG101. Exosomes were quantified by BCA assay of total protein content (Thermo Fisher Scientific Life Sciences). Exosomes from control and IL-1β-primed MSCs were designated Exo-MSCs and Exo-MSCsIL−1β, respectively.
Jem 200cx
Manufactured by Hitachi
Sourced in Japan
The JEM-200CX is a transmission electron microscope (TEM) manufactured by Hitachi. It is designed to provide high-resolution imaging of small-scale specimens. The JEM-200CX uses an electron beam to illuminate and magnify the sample, allowing for detailed analysis of its structure and composition at the nanoscale level.
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3 protocols using jem 200cx
1
Characterization of Exosomes from IL-1β-Primed MSCs
2
Characterization of GNPs and 5-ALA-GNPs
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The morphology of GNPs and 5-ALA-GNPs were investigated via high-resolution transmission electron microscopy (TEM; JEM-200CX, Hitachi, Japan). The diameter of the GNPs and the 5-ALA-GNPs were measured using a ZetaSizer Nano ZS90 instrument (Malvern Instruments, UK). The UV-Vis absorption spectra of GNPs and 5-ALA-GNPs were examined using an ultraviolet-visible spectrophotometer (DU-64, Jasco, Japan).
3
Conjugation of 5-ALA and PpIX with GNPs
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5-ALA and PpIX conjugated with GNPs through electrostatic interaction between positive GNPs and negative 5-ALA and PpIX under the alkalinity environment. 5-ALA-GNPS and PpIX-GNP conjugates were prepared by mixing 500 μl 0.1 nM cationic GNPs with 500 μl 4 mM 5-ALA (Sigma, USA) and 2 mM PpIX (Sigma, USA) in HEPES buffer (pH 7.4, Sigma, USA) rapidly at room temperature. The cationic GNPs were synthesized by mixing poly (diallyldimethylammonium chloride) (PDDA, sigma) and HAuCl 4 (Sigma, USA), and then the mixture was heated with a microwave. In order to adjust the size of the GNPs, we used three different ratios of PDDA and HAuCl 4 for the reaction (Table 1).
The colors of the resultant GNPs and 5-ALA-GNPS conjugate were both light amaranths red. 26, (link)38, (link)39 (link) The absorption spectra of GNPs and 5-ALA-GNPSs were measured on a DU-640 UV-Visible (Vis) spectrophotometer (JASCO, Japan). The conjugates were confirmed by using a transmission electron microscopy (JEM-200CX, HITACHI, Japan) and a particulate size description analyzer (Mastersizer 2000, Malvern, England).
The colors of the resultant GNPs and 5-ALA-GNPS conjugate were both light amaranths red. 26, (link)38, (link)39 (link) The absorption spectra of GNPs and 5-ALA-GNPSs were measured on a DU-640 UV-Visible (Vis) spectrophotometer (JASCO, Japan). The conjugates were confirmed by using a transmission electron microscopy (JEM-200CX, HITACHI, Japan) and a particulate size description analyzer (Mastersizer 2000, Malvern, England).
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