Aichi2 virus was labelled with DiOC18 (Molecular Probes) and R18 using the same protocol described above for R18 labelling except that a probe mixture (6 μl) containing both DiOC18 (33 μM) and R18 (67 μM) was used instead of R18 alone. A549 cells cultured in glass dishes were incubated with fluorescently labelled viruses in the absence or presence of zanamivir (5 μM) for 30 min at 37 °C and then washed with PBS. Imaging was conducted using the dual wavelength imaging technique31 (link). Briefly, the dish was placed on the stage of a confocal microscope (TCS-SP2 MP, Leica, Wetzlar, Germany) equipped with a 100× (NA = 1.4) objective lens. Cells were scanned using an argon-krypton laser (488 nm), and two ranges of emitted light (510–525 nm, green fluorescence; 575–640 nm, red fluorescence) were simultaneously detected before the acquired images were merged.
Dioc18
Manufactured by Thermo Fisher Scientific
Sourced in United States
DiOC18 is a fluorescent dye used for staining and labeling lipophilic structures in cells. It selectively stains the endoplasmic reticulum and Golgi apparatus, providing a useful tool for visualizing these organelles.
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Lab products found in correlation
6 protocols using dioc18
1
Visualizing Viral Dynamics with Dual-Labeled Viruses
2
Electroformation of Giant Unilamellar Vesicles
3
pBMSC Labeling with DiOC18 Dye
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Before injection, pBMSCs (20 × 106 cells/mL) were incubated in 10 μg/mL of DiOC18 (3, 3′-dioctadecyloxacarbocyanine perchlorate) solution (Molecular Probes, part of Life Technologies) containing 3% FCS for 20 minutes at 37°C [27 (link)]. Finally, pBMSCs were washed with 1X Hanks’ balanced salt solution (PAA) three times to get rid of dye remnant and re-suspended in 5% human serum albumin (Albunorm; Octapharma).
4
Systemic Transplantation of Labeled mADSC in Col7a1−/− Neonates
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All animal procedures were performed in accordance with the Guide for the Care and Use of Laboratory Animals (National Institutes of Health publication no. 86-23) and approved by the Institutional Animal Care and Use Committee of the Thomas Jefferson University. For all transplantation studies, mADSC (passages 2–4) isolated from wild-type C57BL/6 J were labeled with a red lipophilic tracer DiOC18 (Molecular Probes, Grand Island, NY, USA) as we described previously [6 (link)]. For systemic transplantation, the 1–2-day-old Col7a1–/– neonates (n = 5/time point) received 0.6 × 106–0.8 × 106 cells in 10–15 μl PBS per mouse intraperitoneally. For direct viewing, transplanted cells were detected using an IVIS live-imaging system (IVIS Lumina XR; Caliper LifeSciences, Alameda, CA, USA). To avoid detection of autofluorescence, the exposure time of all imaged animals was minimized to a maximum of 1 second.
5
Phagocytosis of Fluorescent E. coli
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DioC18 (0.25 mg/mL; Invitrogen, Waltham, MA, USA) labeled Escherichia coli (ATCC 25922) was suspended in 0.5 mL of Hank’s balanced salt solution (HBSS) and used for the analysis of phagocytosis. Granulocytes of 1 × 106 each were preseeded in a 96-well plate and then cocultured with fluorescently labeled bacteria at 1 × 107 DioC18-labeled E. coli. in a PBS solution at 37 °C for 90 min. By the end of the coincubation, 100 μL of trypan blue (1.25 mg/mL) were added to quench the residual DioC18-labeled E. coli. Phagocytosis of the granulocytes was determined by flow cytometry (Becton Dickinson FACSCaliburTM, Franklin Lakes, NJ, USA).
6
Visualizing EV Internalization in hMSCs
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To determine the internalization of Osteo- and Naïve-EVs by the hMSC, 10 µg of Osteo- and Naïve-EVs, respectively, was labelled with a green fluorescent lipophilic dye- 3 mM DiO'; DiOC18 [3 (link)] (3,3′-Dioctadecyloxacarbocyanine Perchlorate/DMSO (DiOC18, Invitrogen™) for 2 h at 37 °C by gentle rotation [29 (link)]. Non-binding DiOC18 dye was then removed by diluting samples with PBS, followed by centrifugation using Vivaspin® 2, 100 kDa MWCO Polyethersulfone filters (Sartorius Stedim Biotech GmbH) at 300 × g for 3 min at 4 °C. hMSC was then washed twice with PBS and incubated at 37 °C with labelled EVs for 48 h, before fixing with 4% paraformaldehyde for 20 min. For staining of filamentous actin (F-actin), phalloidin–tetramethylrhodamine B isothiocyanate (Sigma-Aldrich, St. Louis, MO, USA) was added and incubated for 20 min at room temperature. Cellular nuclear DNA was then stained with 4′,6-diamidino-2-phenylindole (DAPI) for an additional 20 min. The cellular uptake of EVs was imaged using a Dragonfly 505 confocal spinning disc system (Andor Technologies, Inc, Belfast, Northern Ireland).
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