HK-2 cells and HMC were cocultured by Transwell Permeable Support System of 12-well plates (0.4 µm pore-size,Corning, USA),.The former one was seeded in upper chamber while the recipient HMC were plated in the lower chambers. The cells were starved by serum-free medium for 12 h. Afterwards, HK-2 cells were labeled with 10 µM DiO dye (Beyotime, Shanghai, China) in the dark for 30 min. The cocultured experiment was carried out with or without ZWTS and GW4869 for 24 h. A confocal microscope (Zeiss, Germany) was managed to observe the uptake of DiO-labeled exosomes.
Dio dye
Manufactured by Beyotime
Sourced in China
DiO dye is a fluorescent stain used for labeling cell membranes. It is a lipophilic carbocyanine dye that inserts into the lipid bilayer of cell membranes, allowing for the visualization of cellular structures under fluorescence microscopy.
Automatically generated - may contain errors
Lab products found in correlation
Dapi dye, by Beyotime (2 mentions)
Confocal microscope, by Zeiss (1 mentions)
Gw4869, by Merck Group (1 mentions)
Confocal laser scanning microscopy, by Nikon (1 mentions)
15 mm glass bottom cell culture dish, by NEST Biotechnology (1 mentions)
Reactive oxygen species assay kit, by Beyotime (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
Tween 80, by Merck Group (1 mentions)
Span 80, by Merck Group (1 mentions)
Sodium alginate, by Merck Group (1 mentions)
Paraquat methyl viologen dichloride, by Merck Group (1 mentions)
Dii dye, by Beyotime (1 mentions)
Methylene blue, by Merck Group (1 mentions)
1 2 distearoyl sn glycero 3 phosphoethanolamine n methoxy polyethylene glycol 2000 dspe peg2000, by Avanti Polar Lipids (1 mentions)
1 2 dipalmitoyl sn glycero 3 phosphocholine, by Avanti Polar Lipids (1 mentions)
Hoechst 33342, by Beyotime (1 mentions)
Tetraethyl orthosilicate teos, by Merck Group (1 mentions)
Lsm710 fluorescence microscope, by Zeiss (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Abcam (1 mentions)
12 well culture plate, by Corning (1 mentions)
11 protocols using dio dye
1
Exosome Uptake in Cocultured Cells
2
Phagocytosis Assay of Cancer Cells by Macrophages
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M2 type RAW264.7 cells (2 × 105 cells/well) seeded in the confocal petri dish were separately incubated with R848, PR@RM, and PR@RM-M2 for 24 h. Then, they were collected and labeled with DiO dye (Beyotime). In the meantime, 4T1 cancer cells were labeled with Hoechst and co-culture with the above DiO labeled RAW264.7 cells at the number ratio of 1:1. After 24 h, the phagocytosis of 4T1 cells by RAW264.7 cells was observed by CLSM (Zeiss).
3
PTEC-Derived EV Influence on Fibroblasts
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To demonstrate the effect of PTEC-derived EVs on fibroblasts, we established coculture systems with Transwells with 0.4 mm pores (Corning) that mimic the milieu where PTECs are exposed to aldosterone on the apical surface as described before. PTECs were labeled with Dio dye (5 mg/mL, Beyotime) at 37°C for 30 min. After staining, the cells were washed completely three times with PBS to remove free dye. Then, PTECs were grown to complete confluence and cocultured with fibroblasts, which were seeded in the lower compartment of the Transwell system. PTECs were then stimulated on the apical side, and GW4869 (D1692, Sigma) was used as an EV secretion inhibitor. EVs from Dio-labeled PTECs were released on the basolateral side and were internalized by fibroblasts, which was observed using a Leica microscope (Germany).
4
EEC Uptake of DiO-Labeled AMEVs
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The uptake of AMEVs by EECs was performed as described previously.18 (link) Briefly, AMEV pellets (30 μL, approximately 8.04 × 1010±1.128 × 1010 particles) were incubated with a DiO dye (Beyotime Biotechnology, China) solution (10 μM) for 30 min at 37 °C. After washing twice with PBS, pellets were centrifuged at 100,000×g for 90 min, and DiO-AMEVs were resuspended in 100 μL of PBS. EECs (1 × 106/mL) were incubated with DAPI dye (Beyotime Biotechnology, China) (10 μg/mL) in a 15 mm glass bottom cell culture dish (NEST, Hong Kong, China) at 37 °C for 24 h, then washed three times with PBS and treated with DMEM containing 10 μL of DiO-AMEVs for 0 h, 48 h, and 72 h. Images were obtained using laser scanning confocal microscopy (Nikon Microsystems, Japan). Digital images were recorded and analyzed using NIS 4.2 Viewer (Nikon Microsystems, Japan).
5
Phagocytosis Assay of Cancer Cells by Macrophages
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M2 type RAW264.7 cells (2 × 105 cells/well) seeded in the confocal petri dish were separately incubated with R848, PR@RM, and PR@RM-M2 for 24 h. Then, they were collected and labeled with DiO dye (Beyotime). In the meantime, 4T1 cancer cells were labeled with Hoechst and co-culture with the above DiO labeled RAW264.7 cells at the number ratio of 1:1. After 24 h, the phagocytosis of 4T1 cells by RAW264.7 cells was observed by CLSM (Zeiss).
6
Quercetin Nanoparticle Synthesis and Characterization
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Quercetin dihydrate was obtained from Sangon Biological Engineering Technology & Services Co., Ltd. Sodium alginate, Span 80, Tween 80, DAPI and paraquat (methyl viologen dichloride) were purchased from Sigma-Aldrich Co. (St. Louis, USA). Calcium chloride was purchased from Tianjin Fengchuan chemical reagent technologies Co., Ltd. DPPH (1, 1-diphenyl-2-picrylhydrazyl) was obtained from Alfa Aesar (Ward Hill, MA, USA). The interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), and interleukin-6 (IL-6) ELISA kits were purchased from Peprotech Inc. (Rocky Hill, New Jersey, USA). Antioxidant capacity assay kit with FRAP method, Malonaldehyde (MDA) and Catalase (CAT) kits, Reactive Oxygen Species (ROS) assay kit, DiI dye, DiO dye and DAPI dye were obtained from the Beyotime Biotechnology (Shanghai, China).
7
Multifunctional Nanobubbles for Biomedical Applications
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We obtained methylene blue (MB) and tetraethyl orthosilicate (TEOS) from Sigma-Aldrich (St. Louis, MO, USA), while the ethanol, chloroform, and ammonia–water solution were procured from Fuyu Chemical Co., Ltd (Tianjin, China). The 1.2-distearoyl-sn-glycero-3-phosphoethanolamine-N- (methoxy (polyethylene glycol)-2000) (DSPE-PEG 2000) and 1.2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) were purchased from Avanti Polar-lipids, Inc. (Alabaster, AL, USA), in a powdered form, and were used to fabricate NBs. IR783 were bought from Macklin (Shanghai, China) and the C3F8 gas of purity grade 5N was supplied by Newradar Gas Co., Ltd. (Wuhan, China). The DiO dye and Hoechst 33342 were provided by Beyotime Biotechnology Co., Ltd. (Shanghai, China). All chemical reagents were of analytical grade and were used as received without further purification.
8
Visualizing Vip3Aa Subcellular Localization in Sf9 Cells
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The subcellular localization of Vip3Aa or mutants in Sf9 cells was described in our previous study [28 (link)]. Briefly, Vip3Aa (or mutant proteins) with red fluorescent protein (RFP) was used to treat Sf9 cells for 6 h. After washing with PBS buffer three times, the Sf9 cells were stained with Dio dye (Beyotime, Shanghai, China) at 28 °C in the dark for 45 min. Then, the cells were imaged with a Zeiss LSM710 fluorescence microscope.
9
Exosome Uptake Visualization
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DIO dye (Beyotime) was treated with the exosome suspension of HPDE cells. Then, PANC-1 and Capan-2 cells were incubated with DIO-stained exosomes, followed by immobilized with paraformaldehyde and dyed with DAPI. Exosome uptake was observed using fluorescence microscope.
10
Coculture of DPSCs and MDPC23 cells
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For the DPSC and MDPC23 coculture experiments, DPSCs were seeded in the bottom chambers of transwell plates with a pore size of 0.4 µm (Corning, USA). Then, MDPC23 cells were labeled with DiO dye according to the manufacturer’s instructions (Beyotime, China). Then, the labeled MDPC23 cells were placed into the upper chambers of the same wells and incubated for 24 h or 7 days. The 0.4-µm transwell allowed the free exchange of molecules, including exosomes, between the DPSCs and MDPC23 cells but prevented cell migration. Notably, MDPC23 cells were transfected with lentivirus to achieve the shRNA-mediated silencing of TSC1 (shTSC1, 5′-GACACACAGAATAGCTATG-3′) in order to activate mTORC1 before they were cocultured with DPSCs. After 24 h, DPSCs were harvested and stained with DAPI (Abcam, UK). The fluorescence images were taken by Olympus BX63 (72 dpi × 72 dpi), Cellsens software.
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