Antibodies for TLR4, MCP-1, iNOS, ICAM-1, VCAM-1, p38MAPK, p-p38MAPK, JNK1/2, p-JNK 1/2, ERK1/2, p-ERK1/2, β-actin were used for western blot. Trizol reagent, Revert Aid™ First Strand cDNA Synthesis Kit and Dream Taq™ PCR Master Mix were used for RT-PCR. NF-κB nuclear translocation assay kit, total nitric oxide assay kit and 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate (DiI), were from Beyotime Institute of Biotechnology (Shanghai, China). Positive drug simvastatin (SIM) was obtained from National Institutes for Food and Drug Control (Beijing, China). LEE was obtained from the International Biotechnology Research and Development Center of Shandong University at Weihai using our previous procedure13 (link)).
1 1 dioctadecyl 3 3 3 3 tetramethylindocarbocyanine perchlorate
Manufactured by Beyotime
Sourced in China
1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate is a fluorescent dye used in various lab applications. It is a lipophilic cationic carbocyanine dye that can intercalate into the lipid bilayer of cells and membranes.
Automatically generated - may contain errors
Lab products found in correlation
3 3 dioctadecyloxacarbocyanine perchlorate, by Beyotime (4 mentions)
Lysotracker red dnd 99, by Thermo Fisher Scientific (2 mentions)
Total ros assay kit, by Beyotime (2 mentions)
Amyloid β1 40 human elisa kit, by Elabscience (2 mentions)
Amyloid β1 42 human elisa kit, by Elabscience (2 mentions)
Lysosenser yellow blue dextran, by Thermo Fisher Scientific (2 mentions)
Bca protein assay kit, by Beyotime (2 mentions)
Cell counting kit 8 cck 8, by Beyotime (2 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (2 mentions)
Annexin 5 fluorescein isothiocyanate fitc propidium iodide staining kit, by Beyotime (2 mentions)
Resiquimod r848, by Adooq (2 mentions)
Mouse tumor necrosis factor α tnf α, by ABclonal (2 mentions)
Mouse interferon γ ifn γ enzyme linked immunosorbent assay elisa kits, by ABclonal (2 mentions)
Nuclear dye 4 6 diamidino 2 phenylindole dapi the cell counting kit cck 8, by Beyotime (2 mentions)
Membrane dyes of 3 3 dioctadecyloxacarbocyanine perchlorate dio, by Beyotime (2 mentions)
Pluronic f 127, by Merck Group (2 mentions)
Total nitric oxide assay kit, by Beyotime (1 mentions)
Pma tpa, by Beyotime (1 mentions)
Annexin 5 fluos staining kit, by Roche (1 mentions)
2 4 amidinophenyl 6 indolecarbamidine dihydrochloride dapi, by Beyotime (1 mentions)
13 protocols using 1 1 dioctadecyl 3 3 3 3 tetramethylindocarbocyanine perchlorate
1
Investigating Inflammatory Signaling Pathways
2
Nanoparticle-Mediated Cell Assays
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GA (purity ≥99%), PLGA (molecular weight 40,000), polyvinyl alcohol (PVA), and dichloromethane (DCM) were purchased from Aladdin Industrial Corporation (Shanghai, China). 3,3′-Dioctadecyloxacarbocyanine perchlorate (DiO), 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate (DiI), 2-(4-amidinophenyl)-6-indolecarbamidine dihydrochloride (DAPI), phorbol-12-myristate-13-acetate/12-O-tetradecanoylphorbol 13-acetate (PMA/TPA), and a cell-cycle analysis kit were purchased from Beyotime Institute of Biotechnology (Haimen, China). An annexin V–Fluos staining kit was purchased from Hoffman-La Roche Ltd (Basel, Switzerland).
3
Cell Transfection and Imaging Protocol
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A density of 1.0×105 cells per well were seeded into 24-well plates for 6 h. At 12 h after serum-deprivation, cells were transfected with miR-21 mimic (50 nM), inhibitor (100 nM), or their negative controls for 48 h. Then cells were labeled with non-immunological red fluorescent dye 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate (Dil, Beyotime, China) according to the manufacturer’s protocol. Cells were observed under an inverted fluorescence microscope (Leica, Germany) and digital images were acquired for determination of cell size with CellProfiler software.
4
Amyloid-beta Inhibition and Cell Assays
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Rapamycin (Rapa), coumarin-6 (Cou-6), and okadaic acid (OA) were purchased from Sigma–Aldrich Inc. (St.Louis, MO, USA). Gypenoside XVII (GP-17) was attained from Push Biotechnology Co., Ltd. (Chengdu, China). RP-1 (CAPDTKTQ) and Aβ1–42 were synthesized by Guotai Biotechnology Co., Ltd. (Hefei, China) and Chinese Peptide Co., Ltd. (Hangzhou, China), respectively. 1,1′-Dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate (DiI), 3,3′-dioctadecyloxacarbocyanine perchlorate (DiO), Cell Counting Kit-8 (CCK-8), Total ROS assay kit and BCA Protein Assay Kit were purchased from Beyotime Biotechnology Co., Ltd. (Shanghai, China). Amyloid-β1–40 Human ELISA Kit and Amyloid-β1–42 Human ELISA Kit were purchased from Elabscience Biotechnology Co., Ltd. (Shanghai, China). Lysotracker Red DND-99 and Lysosenser Yellow/Blue dextran were purchased from Thermo Fisher Scientific, Inc. (Eugene, OR, USA). The mouse brain microvascular endothelial cells (bEnd.3), mouse hippocampal neuronal cells (HT22), and murine neonatal microglial cells (BV2) were obtained from the Chinese Academy of Sciences Cell Bank (Shanghai, China). All chemicals used were analytical or reagent grade.
5
Amyloid-beta Inhibition and Cell Assays
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Rapamycin (Rapa), coumarin-6 (Cou-6), and okadaic acid (OA) were purchased from Sigma–Aldrich Inc. (St.Louis, MO, USA). Gypenoside XVII (GP-17) was attained from Push Biotechnology Co., Ltd. (Chengdu, China). RP-1 (CAPDTKTQ) and Aβ1–42 were synthesized by Guotai Biotechnology Co., Ltd. (Hefei, China) and Chinese Peptide Co., Ltd. (Hangzhou, China), respectively. 1,1′-Dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate (DiI), 3,3′-dioctadecyloxacarbocyanine perchlorate (DiO), Cell Counting Kit-8 (CCK-8), Total ROS assay kit and BCA Protein Assay Kit were purchased from Beyotime Biotechnology Co., Ltd. (Shanghai, China). Amyloid-β1–40 Human ELISA Kit and Amyloid-β1–42 Human ELISA Kit were purchased from Elabscience Biotechnology Co., Ltd. (Shanghai, China). Lysotracker Red DND-99 and Lysosenser Yellow/Blue dextran were purchased from Thermo Fisher Scientific, Inc. (Eugene, OR, USA). The mouse brain microvascular endothelial cells (bEnd.3), mouse hippocampal neuronal cells (HT22), and murine neonatal microglial cells (BV2) were obtained from the Chinese Academy of Sciences Cell Bank (Shanghai, China). All chemicals used were analytical or reagent grade.
6
Hyaluronic Acid Nanoparticle Synthesis
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Hyaluronic acid (HA), with a molecular weight (MW) of 3000-1000Da, was purchased from the Shandong Freda Biotechnology Co., Ltd. (Shandong, China). 1-Hydroxy-5-pyrrolidinedione (NHS), 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC), 4-methoxybenzoic acid, octadecylamine, indocyanine green (ICG), Dimethyl sulfoxide (DMSO) were all purchased from Aladdin Reagent Database Inc (Shanghai, China). Polyethylene glycol (PEG) with 2000 molecular weight was purchased from Shanghai Yare Biotechnology Co., Ltd (Shanghai, China). 1,1'-Dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI) was purchased from Beyotime Institute of Biotechnology (Jiangsu, China). All inhibitors used were purchased from MedChemExpress (Monmouth Junction, NJ, USA).
7
Exosome Cellular Uptake and Localization
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Cellular uptake of exosomes was assessed by confocal microcopy [45 (link)]. iPSCs-Exos and MSCs-Exos were labeled with DiI fluorescent dye (1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate; Beyotime, China), which labels the plasma membrane. Briefly, exosomes were incubated with DiI dye for 1 h in the dark. Then labeled-exosomes were resuspended in 4 ml PBS followed by ultracentrifugation at 110,000 g for 1 h. The pellets were resuspended in 100 μl of PBS. HCECs were incubated with labeled exosomes for 24 h and then washed with PBS to remove unbound exosomes. Mounting medium with 4',6-diamidino-2-phenylindole (DAPI; Vector Laboratories, US) was used to stain nuclei, and images were captured under a confocal microscope (Carl Zeiss, Germany).
To examine the uptake of iPSCs/MSCs-Exos by the corneal epithelium in vivo, DiI-stained exosomes were applied topically to the cornea of rats, and assessment occured 24 h later. The cornea was then obtained from rats and stained with DAPI, mounted on a slide, and imaged with confocal microscopy. The corneal epithelium of rats was also analyzed by TEM with the same method described above.
To examine the uptake of iPSCs/MSCs-Exos by the corneal epithelium in vivo, DiI-stained exosomes were applied topically to the cornea of rats, and assessment occured 24 h later. The cornea was then obtained from rats and stained with DAPI, mounted on a slide, and imaged with confocal microscopy. The corneal epithelium of rats was also analyzed by TEM with the same method described above.
8
Multimodal Imaging and Molecular Regulation
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3,3′-dioctadecyloxacarbocyanine perchlorate
(DiO), 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine
perchlorate
(DiI), 1,1'-Dioctadecyl-3,3,3',3'-Tetramethylindodicarbocyanine,4-Chlorobenzenesulfonate
Salt(DiD), ROS flow kit, and Western blot reagents were purchased
from Beyotime Biotechnology (China). The cytokines used in this study
(TNF-α, IL-1β, IL-6, interferon-γ (IFN-γ)),
lipopolysaccharide (LPS), and NGF were purchased from MedChemExpress
(MCE, China). Indocyanine green (ICG) was obtained from Macklin Inc.
(China). The apoptosis flow kit and cell counting kit-8 (CCK-8) cell
viability assay kit were purchased from Vazyme Biotech Co., Ltd. (China).
The J774A.1 cell line was purchased from Procell Life Science &
Technology Co., Ltd. (China). Lipofectamine 3000, PBS, media, serum,
and ELISA kits required for cell culture were purchased from Thermo
Fisher Scientific Inc. (Waltham, MA, USA). The PCR-related reagents
were purchased from Yeasen Biotechnology Co., Ltd. (China). The animals
needed for the experiment were purchased from the Laboratory Animal
Center of Huazhong University of Science and Technology.
(DiO), 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine
perchlorate
(DiI), 1,1'-Dioctadecyl-3,3,3',3'-Tetramethylindodicarbocyanine,4-Chlorobenzenesulfonate
Salt(DiD), ROS flow kit, and Western blot reagents were purchased
from Beyotime Biotechnology (China). The cytokines used in this study
(TNF-α, IL-1β, IL-6, interferon-γ (IFN-γ)),
lipopolysaccharide (LPS), and NGF were purchased from MedChemExpress
(MCE, China). Indocyanine green (ICG) was obtained from Macklin Inc.
(China). The apoptosis flow kit and cell counting kit-8 (CCK-8) cell
viability assay kit were purchased from Vazyme Biotech Co., Ltd. (China).
The J774A.1 cell line was purchased from Procell Life Science &
Technology Co., Ltd. (China). Lipofectamine 3000, PBS, media, serum,
and ELISA kits required for cell culture were purchased from Thermo
Fisher Scientific Inc. (Waltham, MA, USA). The PCR-related reagents
were purchased from Yeasen Biotechnology Co., Ltd. (China). The animals
needed for the experiment were purchased from the Laboratory Animal
Center of Huazhong University of Science and Technology.
9
Nanoparticle-based Drug Delivery System
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Mulberry leaf and Pueraria Lobata were purchased from Tongrentang drugstore. Reduced l-glutathione, Na2SeO3 and Poloxamer188 were from Aladdin Reagent (Shanghai, China). PLGA10,000–PEG2000 (50/50) and 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) were provided by RESENBio (Xi’an, China). Rutin and puerarin were purchased from Ruifensi Bio-Tech (Chengdu, China). Simvastatin, chlorpromazine, filipin, latrunculin B and ethylisopropylamiloride (EIPA) were purchased from Sigma–Aldrich (MO, USA). 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate (DiI) was obtained from Beyotime (Shanghai, China). HPLC-grade acetonitrile was provided by Merck Corp. (Darmstadt, Germany). All other chemicals were of analytical grade and used as received.
10
Quantifying ACE2 Membrane Localization
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A549-ACE2 and Huh-7 cells were plated in six-well plates, and we treated the cells with palbociclib, mimosine or serum starvation for cell cycle arrest for 48h. After washing cells three times with PBS, cell membranes were stained with fluorescent probe Dil (1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate (Beyotime, Shanghai, China). Then cells were washed and fixed in 4% paraformaldehyde and blocked with PBS containing 1% BSA. Cells were incubated overnight with primary antibody ACE2 (AF933, R&D Systems) at 4 °C. The corresponding secondary antibody Donkey anti-Goat IgG (H + L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 488 (Invitrogen, A-11055) was used for incubation for 1h at room temperature in dark. Subsequently, cells were counterstained with DAPI (Beyotime, Shanghai, China) at room temperature and immunofluorescence signals were detected using Stimulated emission depletion (STED) confocal microscope (Leica TCS SP8 gSTED 3X, Leica, Germany). The mean intensity of ACE2 was evaluated by LAS X software. Mander's overlap coefficient (MOC) was used to evaluate ACE2 co-localized with the cell membrane as a fraction of the total ACE2 protein by Coloc 2 in Image J, in which the Manders' tM2 (Above autothreshold) was obtained.
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