DIR (Thermo Fisher, D12731) is a near IR fluorescent, lipophilic dye, quite photostable when incorporated into membranes: once applied to cells, the dye diffuses within the plasma membrane. This dye is useful for in vivo cell tracking; however, we initially tested its toxicity in vitro. To evaluate the highest non-toxic concentration, cells were labeled with DIR. At two different final concentrations (5 and 2.5 µM). 1.0 × 105 cells/sample were incubated for 20 min with DIR at 37 °C in the dark. When the incubation time ended, cells were washed with PBS and counted with Trypan Blue (Sigma, T8154) to measure cell viability. The final concentration of 2.5 µM was established as non-toxic for HepaRG cells.
D12731
Manufactured by Thermo Fisher Scientific
Sourced in United States
The D12731 is a laboratory instrument designed for conducting biochemical analyses. It is a high-performance device that can perform various analytical techniques. The core function of the D12731 is to facilitate the measurement and detection of specific analytes in samples.
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Lab products found in correlation
14 protocols using d12731
1
Evaluation of DIR Dye Toxicity
2
Tracking HucMSCs Biodistribution Post-MI
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HucMSCs were pre-stained with the near-infrared fluorescent-lipophilic tracer 1,1-dioctadecyl-3,3,3,3-tetramethylindotricarbocyanine iodide (DiR, ThermoFisher, D12731) according to the manufacturer’s instructions, and then 3*105 DiR-labeled HucMSCs were injected in the heart of mice after MI operation. The fluorescence intensity of multiple organs (including heart, lung, liver, kidney and spleen) was analyzed using the IVIS imaging instrument (Xenogen, USA) with the relevant channel.
3
Fluorescent Labeling of Extracellular Vesicles
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Freshly isolated EVs were incubated with either 1 μM of the fluorescent lipophilic tracer DiR (D12731, LifeTechnologies) or 1 μM of DiD (D7757, LifeTechnologies) at 4 °C for 30 min. 100 μL of each sample were placed on top of a size exclusion column (Exo-spin®) and centrifuged at 50×g for 1 min. 200 µL of PBS were then placed on top of the column and the EVs were obtained after centrifugation at 50×g for 1 min.
4
In Vivo Tracking of Exosome Uptake
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Exo-MS was incubated with 1 mM fluorescent lipophilic tracer DiR (1, 1-octadecyl-3,3,3, 3-tetramethylindotricarbamine iodide) (Invitrogen, D12731, Life Technologies) at room temperature in the dark for 15 minutes. The exosomes were then separated at 120000g×70 min and the excess dye was washed. Exo-MS with a concentration of 100μg/100μL/mice was injected into the anesthetized mice via the tail vein. In vivo imaging was performed 24 h after injection, after which the mice were sacrificed and the relevant organs were removed. In the control group, 100ul PBS was injected into the tail vein and observed 24 hours later. DiR-EVs distribution was analyzed using an IVIS Spectrum system (Perkin Elmer).24 (link)
5
In vivo EV Biodistribution Imaging
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EVs were isolated from HEK293T cell cultures by TFF/BE‐SEC as described above and stored for 2 months in PBS‐HAT buffer at ‐80˚C. After thawing, 2 × 1012 EVs were labelled with DiR (1,1‐dioctadecyl‐3,3,3,3‐tetramethylindotricarbocyanine iodide, cat D12731, Life Technologies) O/N at 4°C in a total volume of 2 ml as described previously (Wiklander et al., 2015 (link)). In brief, EVs were injected intra‐venously (tail vein) at doses of 2 × 1011 EVs in a volume of 100 μl per mouse (n = 3 C57BL/6 mice). Organs were analysed with IVIS 6 h post injection ex vivo. The animal experiments were approved by the Swedish Local Board for Laboratory Animals. The experiments were performed in accordance with the ethical permissions granted and designed to minimize the suffering and pain of the animals.
6
In Vivo Tracking of Labeled ONVs
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ONVs were pelled in the presence of 1 μM fluorescent lipophilic tracer DiR (1,1-dioctadecyl-3,3,3,3-tetramethylindotricarbocyanine iodide) (D12731, Invitrogen) by ultracentrifugation (110,000 × g, 90 min). Mice received either 150 μg of DIR-labeled ONVs58 (link) or mock-PBS-DiR as control through gavage. Six hours after gavage, animals were sacrificed and the organs were carefully harvested and imaged for 2 s (excitation, 710 nm; emission, 760 nm) using a high sensitivity charge-coupled device (CCD) camera in vivo imaging system (IVIS) Spectrum (PerkinElmer, USA). Data were normalized on mock-PBS-DiR. Experiments were approved by the Swedish Local Board for laboratory animals and were performed in accordance with ethics permission and designed to minimize animal suffering and pain.
7
Tracking HF-MSCs In Vivo Using DiR Dye
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DiR is a lipophilic fluorescent dye that can be used to stain cell membranes. DiR (D12731; Invitrogen) labeled HF-MSCs were used to track HF-MSCs in vivo. Adjust the third-generation HF-MSCs concentration to 1 × 106/L, and add 5 uL of three mmol/L DIR storage solution to one mL of cell suspension. The DiR solution and cells were incubated for 30 min. Then the stained cells were transplanted via tail vein. After 48 h, the biodistribution of DiR-labeled HF-MSCs were performed by in vivo imaging System (Night OWL II LB983).
8
Evaluation of Modified Nanoparticles Uptake
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NP was labeled with DIR (1,1-dioctadecy 1–3,3,3,3-tetramethy-lindotricarbocyanine iodide) (D12731, Invitrogen, Life Technologies) according to the manufacturer’s protocol. Then DIR-labeled Agomir-122@MNP, Agomir-122@MmNP, and Agomir-122@NP were obtained. Next, the three agents above was separately co-cultured with fibroblasts for 12 h, or with macrophages for 4 h. Culture medium was discarded afterwards and the dish was washed with PBS in triplicate. Cells were fixed in 4% PFA and stained with DAPI for 3 min. Finally, the fluoresecne of labeled agents internalized by fibroblasts was observed microscopically (ECHO Revolve, America), while that by macrophages was measured by flow cytometry.
9
Purification and Labeling of Extracellular Vesicles
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CM was pre-cleared first by a low-speed centrifugation step (500 × g for 10 min) followed by centrifugation at 2,000 × g for 10–20 min to remove larger particles and debris. Unless indicated otherwise, samples were subsequently filtered through a syringe filter (VWR) or bottle top filters (Corning, low protein binding) with cellulose acetate membrane with a 0.22 µm pore size to remove any remaining larger particles. The CM was then ultra-filtrated either using Amicon Ultra-15 100 kDa (Millipore) spin filter at 4000 x g for 30 min or using tangential flow filtration (MicroKross, 20 cm2, SpectrumLabs) with a cut-off of 300 kDa to concentrate the CM. In some experiments, the concentrated retentate was further purified by size exclusion chromatography column (iZON biosciences) according to the manufacturer’s instruction to further purify the EVs.
For DiR labelling, filtered CM was incubated with 1 µM fluorescent lipophilic tracer DiR (1,1-dioctadecyl-3,3,3,3-tetramethylindotricarbocyanine iodide) (D12731, Invitrogen, Life Technologies) at room temperature (RT) for 30 min prior to EV isolation by ultracentrifugation at 120,000 x g for 70 min (Beckman coulter).
For DiR labelling, filtered CM was incubated with 1 µM fluorescent lipophilic tracer DiR (1,1-dioctadecyl-3,3,3,3-tetramethylindotricarbocyanine iodide) (D12731, Invitrogen, Life Technologies) at room temperature (RT) for 30 min prior to EV isolation by ultracentrifugation at 120,000 x g for 70 min (Beckman coulter).
10
Visualizing BMSC-EV Localization in Shoulder Capsule
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To visualize the target tissue (shoulder capsular) of EVs in vivo, BMSC-EVs were labeled with DIR (1,1-dioctadecyl-3,3,3,3-tetramethy-lindotricarbocyanine iodide) (D12731, Invitrogen, Life Technologies) according to the manufacturer's instructions. In general, 100 μg of BMSC-EVs were incubated with DIR (1:1000 in PBS) in the darkness for 15 min, and then the labeled BMSC-EVs were washed in 40 ml PBS, with centrifugation at 120,000 g for 100 min to avoid the excess dye. After then, the DIR-labeled EVs were injected into the shoulder joint cavity of C57/6J mice (8 weeks old, dosage: 100 μg of EVs/150 μl of PBS), and PBS without DIR was used as the control group. After 1h, 3d, or 7d, the mice were subject to in vivo imaging. Finally, fluorescence intensity was evaluated by the IVIS software (Living Image Software for IVIS).
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