Dio dye

Manufactured by Thermo Fisher Scientific

Sourced in United States

DiO dye is a lipophilic, fluorescent probe used to label the plasma membrane of cells. It can be used to visualize cell morphology and facilitate membrane dynamics studies.

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DiO lipophilic dye (8 citations) Fast DiO membrane dye (5 citations) Vybrant DiO dye (4 citations) Vybrant DiI and DiO cell membrane dyes (2 citations) Vybrant DiO lipophilic dye (2 citations) 3,3’-dioctadecyloxacarbocyanine perchlorate (DiO) lipophilic dye (2 citations) Vybrant™ DiO green fluorescent dye (2 citations)

10 protocols using dio dye

Exosome Labeling and Fibroblast Uptake

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ADSC-Exos were labeled with a green, fluorescent 3,3'-dioctadecyloxacarbocyanine perchlorate (DiO) dye (Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. Briefly, 1 µl DiO dye (200 µg/ml; Thermo Fisher Scientific, Inc.) was added to 20 µl exosome suspension and incubated for 20 min at 37˚C. The reaction was stopped by the addition of an equivalent volume of exosome-depleted BSA (Sigma-Aldrich; Merck KGaA), before DiO-labeled exosomes were obtained.
Fibroblasts were washed twice with PBS and seeded at a density of 2x10⁵ into six-well plates for 48 h at 37˚C. Next, 50 ng/µl DiO-labeled exosomes were added to the fibroblasts and incubated at 37˚C in the dark for 6 h before being subsequently fixed with 4% paraformaldehyde at room temperature for 15 min. The cells were then incubated with 0.5% Triton X-100 at room temperature for 15 min and blocked with 10% BSA at 37˚C for 30 min. The fibroblasts were then stained with 2.5% phalloidin (cat. no. 40734ES75; Shanghai Yeasen Biotechnology Co., Ltd.) at room temperature for 20 min. After washing with PBS, the nuclei were then stained with DAPI at room temperature for 3 min (0.5 µg/ml; Invitrogen; Thermo Fisher Scientific, Inc.). The fluorescent cells were visualized using a Zeiss LSM 800 confocal microscope at x100 magnification (Zeiss GmbH).

Liu J., Li F., Liu B., Yao Z., Li L., Liu G., Peng L., Wang Y, & Huang J. (2021). Adipose-derived mesenchymal stem cell exosomes inhibit transforming growth factor-β1-induced collagen synthesis in oral mucosal fibroblasts. Experimental and Therapeutic Medicine, 22(6), 1419.

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Exosome Isolation and Characterization

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Lipofectamine 2000, Dulbecco's modified Eagle's medium (DMEM), α‐minimal essential medium (α‐MEM), Opti‐MEM, fetal bovine serum, horse serum, penicillin‐streptomycin, 0.25% trypsin‐EDTA, DiO dye, Exosome Spin Columns, Micro BCA Protein Assay kit, as well as CD11b, CD86, CD206, CD11c, CD80 monoclonal antibodies were purchased from Thermo Fisher Scientific, USA. Calnexin, CD9, CD63 and CD81, FASL, Caspase‐3, Caspase‐8, and PARP antibodies were purchased from Abcam. 3‐(4, 5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT), RNAlater, dimethyl sulfoxide (DMSO), LysoTracker Green DND‐26, isothiocyanate‐labeled phalloidin and Hoechst 33342 were purchased from Sigma Aldrich, USA. Filter membranes and ultrafilter tubes were purchased from Millipore (Shanghai, China). Cy5‐labeled siRNA (Cy5‐NC), siPLK1 (targeting PLK1), and siPD‐L1 (targeting PD‐L1) were supplied by Suzhou Ribo Life Science Co., Ltd. (Suzhou, China) or Suzhou Biosyntech Co., Ltd (Suzhou, China). All the primers were provided by BioSune Co., Ltd (Shanghai, China). DCFH‐DA, Annexin V‐FITC/PI Apoptosis Detection Kit, and Hieff qPCR SYBR Green Master Mix were purchased from Yeasen (Shanghai, China). Chlorin e6 (Ce6) was purchased from Macklin Biochemical Technology Co., Ltd (Shanghai, China). Optimal cutting temperature (OCT) compound was from Sakura Finetek USA, Inc. (Torracne, CA90501, USA).

Zhang M., Shao W., Yang T., Liu H., Guo S., Zhao D., Weng Y., Liang X, & Huang Y. (2022). Conscription of Immune Cells by Light‐Activatable Silencing NK‐Derived Exosome (LASNEO) for Synergetic Tumor Eradication. Advanced Science, 9(22), 2201135.

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Characterizing EV Isolation and Release

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In order to confirm if the EVs were indeed captured and subsequently released from the separation channel, DiO dye (ThermoFisher Scientific) was flushed through the separation channel with a 10‐min incubation at room temperature. The DiO tagging of EVs to the anti‐CD63 antibody was verified through confocal microscopy examination (A1R, Nikon) showing an increase in fluorescence in the separation channel (Figure 2B(i)). Following elution using the glycine‐HCl buffer the observed fluorescence decreased suggesting successful elution of the captured EVs as seen in Figure 2B(ii). Scanning electron microscopy (SEM) imaging showed not all EV particles passed through the NCAMs (Figures 2C(i) and 2C(ii)) despite being < 200 nm in size.

Casadei L., Choudhury A., Sarchet P., Mohana Sundaram P., Lopez G., Braggio D., Balakirsky G., Pollock R, & Prakash S. (2021). Cross‐flow microfiltration for isolation, selective capture and release of liposarcoma extracellular vesicles. Journal of Extracellular Vesicles, 10(4), e12062.

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EV Isolation and Characterization via LCCM and DiO Tagging

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The DiO dye (ThermoFisher Scientific) was flushed through the separation channel and incubated at room temperature for 10 minutes after the expected capture of the EVs following LCCM perfusion through the injection channel and subsequent isolation of the EVs. The tagging of EVs to the anti-CD63 antibody was verified through confocal microscopy examination (A1R, Nikon). Post DiO-tagging, an increase in fluorescence in the separation channel was observed (Fig. 2B (a)). Following elution using glycine-HCl buffer, fluorescence decreased, suggesting successful elution of EVs (Fig. 2B (b)).
Scanning electron microscope (SEM) imaging showed particle accumulation on the NCAMs suggesting that not all EVs and LCCM particles pass through (Fig. 2C). Based on total volume of fluid collected for each tested flow rate over the fixed 30-minute test, it was noted that only ~4.2% of incoming fluid volume is the permeate across the NCAM and used for isolation of EVs.

Sundaram P.M., Casadei L., Lopez G., Braggio D., Balakirsky G., Pollock R, & Prakash S. (2020). Multi-Layer Micro-Nanofluidic Device for Isolation and Capture of Extracellular Vesicles Derived from Liposarcoma Cell Conditioned Media. Journal of microelectromechanical systems : a joint IEEE and ASME publication on microstructures, microactuators, microsensors, and microsystems, 29(5), 776-782.

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Tracking Liver Uptake of Labeled EVs

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DiO dye (Thermo Fisher) was added to the pellet of EVs isolated from plasma. The EVs were resuspended in PBS and ultracentrifuged twice for 90 minutes at 100,000 g at 4°C. The labeled EVs were resuspended in sterile PBS solution and injected into the portal vein of C57BL/6J mice. Livers were harvested and incubated in modified H69 media at 37°C for 1 hour. The livers were then processed for cryosectioning and immunofluorescence.

Al Suraih M.S., Trussoni C.E., Splinter P.L., LaRusso N.F, & O’Hara S.P. (2020). Senescent Cholangiocytes Release Extracellular Vesicles that Alter Target Cell Phenotype Via the Epidermal Growth Factor Receptor. Liver international : official journal of the International Association for the Study of the Liver, 40(10), 2455-2468.

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Synthesis and Characterization of Lipid-Based Nanocarriers

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DIM-P was synthesized as described previously (32 (link)). The triglycerides Miglyol 812, Compritol 888 ATO, Dynasan 118, Precirol, Gleol, Monosterol, Lebrasol and Transcutol were from Sasol Germany GmbH (Witten, Germany) and Gattefosse (Saint Priest, France). 1,2-dioleoyl-sn-glycero-3-[(N-(5-amino-1-carboxypentyl) imidodiacetic acid) succinyl nickel salt] (DOGS-NTA-Ni) was purchased from Avanti Polar lipids (Alabaster, USA). Fetal bovine serum (FBS), antibiotics mix and lipophilic fluorescent dyes (DIO dye and DID-oil) were from Invitrogen Corp (Eugene, OR). The A549 and H1650 human NSCLC cell line were obtained from American Type Culture Collection (Rockville, MD, USA). Cells were grown in RPMI, F12K/DMEM mediums (Sigma, St. Louis, MO, USA) supplemented with 10% FBS and antibiotic mixture. The cells were maintained at 37°C in the presence of 5% CO2 in air. All other chemicals used in this research were of analytical grade. The six histidine tagged PEGylated YSA (6His-PEG-YSA) tumor homing peptide and control non specific peptide YKA (6His-PEG-YKA) peptide were synthesized by GenScript Corporation (NJ, USA).

Patel A.R., Chougule M, & Singh M. (2014). EphA2 Targeting Pegylated Nanocarrier Drug Delivery System for Treatment of Lung Cancer. Pharmaceutical research, 31(10), 2796-2809.

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Multicolor Cell Coculture and Secretome Analysis

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SH-SY5Y neuron cells and U937 macrophage or HMC3 microglia cells were stained with DID and DIO dye (Invitrogen), respectively, for 30 min after differentiation to facilitate distinguishing in counting. SH-SY5Y neuron cells and U937 macrophage or HMC3 microglia cells (2 × 10⁵ cells·mL⁻¹) were mixed at a 1:1 ratio (vol/vol) and pipetted into the microchip. An antibody-patterned glass slide was imposed and clamped to the microchip to remove the excess cell suspension after being settled for 5 min. After cell culture for 18 h at 37 °C in a 5% CO₂ incubator, secretions were analyzed via immunofluorescence assay. Briefly, the antibody-patterned glass slide was disassembled carefully and blocked with 3% BSA for 1 h. A mixture solution containing the biotin-labeled detection antibodies (for details, see SI Appendix, Table S2) was added and incubated for 45 min to finish the immunoreaction. Then the streptavidin-labeled APC was added for another 30 min incubation. The glass slide was washed using DPBS, 50/50 DPBS/DI water, and DI water sequentially, followed by rinsing and drying, and then it was scanned using the GenePix 4300A scanner (Molecular Devices).

Deng J., Ji Y., Zhu F., Liu L., Li L., Bai X., Li H., Liu X., Luo Y., Lin B, & Lu Y. (2022). Mapping secretome-mediated interaction between paired neuron–macrophage single cells. Proceedings of the National Academy of Sciences of the United States of America, 119(44), e2200944119.

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Visualizing Virus Entry Inhibition by 8P9R

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To identify the effect of 8P9R on virus, H1N1 virus or SARS-CoV-2 was pre-labelled by green Dio dye (Invitrogen, Cat^#3898) according to the manufacture introduction. Dio-labeled virus was treated by 8P9R, P9RS, or P9R (25 μg ml⁻¹) for 45 min. MDCK or Calu-3 cells were infected by the pre-treated virus for 1 h. Virus and cells were fixed by 4% formalin. Cell membrane was stained by membrane dye Alexa 594 (red, Invitrogen, W11262) and cell nucleus were stained by DAPI (blue). Virus entry or without entry on cell membrane was determined by confocal microscope (Carl Zeiss LSM 700, Germany).

Zhao H., To K.K., Lam H., Zhou X., Chan J.F., Peng Z., Lee A.C., Cai J., Chan W.M., Ip J.D., Chan C.C., Yeung M.L., Zhang A.J., Chu A.W., Jiang S, & Yuen K.Y. (2021). Cross-linking peptide and repurposed drugs inhibit both entry pathways of SARS-CoV-2. Nature Communications, 12, 1517.

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Fluorescent Labeling and Biodistribution of F. alocis EVs

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F. alocis EVs (500 μg of protein/1 ml PBS) were stained with Vybrant DiO dye (5 μl of 1 mM DiO dye, Invitrogen; cat#: V22886) at 37°C for 30 min. To exclude residual DiO dye, the DiO dye‐labeled F. alocis EV solutions were filtered using a 100‐kDa centrifugal filter. The retentate containing EVs were resuspended in PBS and concentrated using ultracentrifugation (160,000 × g) at 4°C for 1 h. Then, DiO dye‐labeled F. alocis EVs were resuspended in 1 ml of PBS. No pellets were visible when the same protocol was applied to the DiO dye solution without F. alocis EV. Eight‐week‐old male mice were intraperitoneally administered 50 μg of DiO dye‐labeled F. alocis EVs. At 3 or 12 h, the mice were euthanized, and the organs were collected and analysed within 5 min. The fluorescence intensity in the organs was visualized by a Fusion‐FX6 imaging system (Vilber Lourmat, Marne‐la‐Vallée, France).

Kim H.Y., Song M., Gho Y.S., Kim H, & Choi B. (2021). Extracellular vesicles derived from the periodontal pathogen Filifactor alocis induce systemic bone loss through Toll‐like receptor 2. Journal of Extracellular Vesicles, 10(12), e12157.

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Visualizing Influenza Virus Binding and Entry

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H1N1 virus was labeled by green Dio dye (Invitrogen, Cat^#3898) according to the manufacture introduction. DIO-labeled virus was treated by TAMRA-labeled P9R and TAMRA-labeled P9RS for 1 h at room temperature. Pre-cool MDCK cells were infected by the peptide-treated virus on ice for 15 min and then moved to 37 °C for incubation for 15 min. Cells were washed twice by PBS and then fixed by 4% formalin for 1 h. Nuclei were stained by DAPI for taking images by confocal microscope (Carl Zeiss LSM 700, Germany).

Zhao H., To K.K., Sze K.H., Yung T.T., Bian M., Lam H., Yeung M.L., Li C., Chu H, & Yuen K.Y. (2020). A broad-spectrum virus- and host-targeting peptide against respiratory viruses including influenza virus and SARS-CoV-2. Nature Communications, 11, 4252.

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