Purified EVs were stained with 10−6 M PKH26 or PKH67 Cell Membrane Labeling Dye (Sigma–Aldrich, St. Louis, MO) according to the manufacturer's instructions. Excess dye was removed using exosome spin columns (MW 3000; ThermoFisher Scientific, Carlsbad, CA) per the manufacturer's instructions. Labeled EVs were added to the recipient cells (105 EVs per cells) and incubated for 24 hours. After treatment, cells were washed twice with PBS and changed to fresh medium. Fluorescence intensity was measured with a Celigo high‐throughput micro‐well image cytometer (Nexcelom Bioscience, Lawrence, MA) using its analysis program. The parameters of data acquisition and analysis were set as follows: intensity threshold (4), precision (high), cell diameter (20), dilation radius (0 pixel), cell intensity range (0–255), and cell area (10–10,000 in setting).
Mw 3000
Manufactured by Thermo Fisher Scientific
Sourced in United States
The MW 3000 is a laboratory instrument designed for molecular weight determination. It utilizes advanced technology to precisely measure the molecular weights of a variety of samples, including polymers, proteins, and other macromolecules. The core function of the MW 3000 is to provide accurate and reliable molecular weight analysis data to support research and development activities in various scientific fields.
Automatically generated - may contain errors
Lab products found in correlation
Ivis spectrum system, by PerkinElmer (3 mentions)
Ms 222, by Merck Group (2 mentions)
Opti mem, by Thermo Fisher Scientific (2 mentions)
Syto rnaselect green fluorescent cell stain, by Thermo Fisher Scientific (2 mentions)
Calcein am, by Thermo Fisher Scientific (2 mentions)
Lsm 880, by Zeiss (2 mentions)
Pkh26, by Merck Group (1 mentions)
Celigo high throughput micro well image cytometer, by Revvity (1 mentions)
Celltracker deep red dye, by Thermo Fisher Scientific (1 mentions)
Cmtmr, by Thermo Fisher Scientific (1 mentions)
Prolong gold antifade mounting medium, by Thermo Fisher Scientific (1 mentions)
Cm3050s cryostat, by Leica (1 mentions)
Ami htx, by Spectral Instruments Imaging (1 mentions)
Facscalibur flow cytometer, by FlowJo (1 mentions)
Pkh67 fluorescent cell linker kit, by Merck Group (1 mentions)
Fv1000, by Olympus (1 mentions)
Cm a954, by Thermo Fisher Scientific (1 mentions)
Allegra x 15r, by Beckman Coulter (1 mentions)
Pd 10 column, by GE Healthcare (1 mentions)
Bodipy tr ceramide, by Thermo Fisher Scientific (1 mentions)
27 protocols using mw 3000
1
Labeling and Tracking Extracellular Vesicles
2
DEX Labeling and Organ Imaging
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DEXs suspended in PBS (Hyclone; Cytiva) were labeled with a 1:400 dilution of DiR (Thermo Fisher Scientific) for 30 min at 37 °C. Excess dye was then removed and the DEXs purified using exosome spin columns (MW 3000; Thermo Fisher Scientific). Near-IR fluorescence images were then acquired using the IVIS Spectrum System (PerkinElmer). We used this system to compare the maximal fluorescence intensity of the body and main organs between the DEXs and DEXs-Gel groups.
3
Tracking Exosome Uptake in Cardiomyocytes
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Isolated exosomes were incubated with 3.3 μL of Alexa FlourTM 488 C5 Maleimide (200 μg/mL, A10254, Thermo Scientific, San Jose, CA, USA) for 1 h at room temperature. The labelling was disturbed by passing through the exosome spin column (MW3000, 4484449, Thermo Scientific, San Jose, CA, USA), according to manufacturer’s instruction. The labelled exosomes were washed out and resuspended with 1 mL of serum free OptiMEM (31985088, Thermo Scientific, San Jose, CA, USA). For each well in a 4-well plate, 250 μL labelled exosomes were incubated with primary cardiomyocytes in the standard cell culture condition for 4 h at 37 °C. Cardiomyocytes were then counterstained with CellTracker Deep Red dye and mounted with ProLong Gold antifade mountants without DAPI (#P36934, Thermo Scientific, San Jose, CA, USA). The cells co-labelling with Maleimide (green) and cell tracker (red) under the confocal microscope were considered as positive cells containing exosomes.
4
Retrograde Tracing of Motoneurons and DRG Neurons
5
Tracking Apoptosis in Cancer Spheroids
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EVs (20 µg) were stained with 10 µM CMTMR (ThermoFisher Scientific), and washed using exosome spin columns (MW3000, ThermoFisher Scientific). HCT116 spheroids were incubated with CMTMR-stained EVs and 12 µM of CellEvent Caspase-3/7 Green Detection Reagent for 3 days. Single tumor spheroids were fixed with 4% formaldehyde for 1 h, aligned on the bottom of a plastic mold and snap-frozen in OCT compound (Tissue-Tek) using liquid nitrogen. Cryosections of 10 µm, with 20 µm intervals, were obtained using a Leica cm3050s cryostat. Sections were stained with DAPI and embedded in Prolong Gold antifade mounting medium (ThermoFisher) and analyzed using Olympus fluorescence microscope.
6
Retrograde Tracing of Motoneurons and DRG Neurons
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Motoneurons and DRG neurons were back-labeled by injecting dextran dyes (Tetramethylrhodamine-dextran, MW 3000, Thermo Fisher, Cat# D3308; Alexa Fluor 647-dextran, MW 10000, Thermo Fisher Cat# D22914) into muscles. Crystals of the dyes were first dissolved in distilled water and subsequently desiccated on glass slides. Tg(Chx10:GFP) zebrafish were anaesthetized in 0.01% tricaine methanesulfonate (MS-222, Sigma-Aldrich) and placed lateral side up in a Petri dish. The tracers were injected with the tip of sharp tungsten pins in a selective quadrant of the fast axial musculature or in the full axial musculature (Figure S1 ). Animals were allowed to recover for at least 2 h prior to electrophysiology experiments. For post-hoc morphology analysis single V2a INs and pMNs were passively filled through the recording electrode with 0.25% neurobiotin. For dye-coupling experiments, one pMN was passively filled through the recording electrode with 2% neurobiotin.
7
Near-IR Imaging of Labeled DEX
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We used DiR (Thermo Fisher Scientific, Waltham, MA, MA, USA) for labeling DEX. Then, we used exosome spin columns (MW 3000; Thermo Fisher Scientific, Waltham, MA, USA) to remove the dye and purify DEX. Finally, we used the IVIS Spectrum System (PerkinElmer, Houston, TX, USA) to acquire the near-IR fluorescence images and compare the radiant efficiency of the body, spleen, and liver among different groups.
8
Biodistribution of Labeled sEVs
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sEVs suspended in PBS were labeled with 1:400 diluted DiR (Thermo Fisher Scientific) for 30 min at 37°C and dye was removed to purify sEVs using exosome spin columns (MW 3000; Thermo Fisher Scientific). The rats were sacrificed, and the heart, liver, spleen, lungs, and kidneys were acquired to examine the distribution of sEVs in various major organs. Near-IR fluorescence images were acquired using the IVIS Spectrum System (PerkinElmer), and fluorescence intensity of main organs between the sEVs and sEVs-Gel groups was compared.
9
EV-endMSCs Uptake by Murine Embryos
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To analyze the uptake of the EV-endMSCs by the embryos, the extracellular vesicles were stained with 2% SYTO RNA Select green fluorescent cell stain (Thermo Fisher Scientific) at 37°C for 30 min. PBS without exosomes were stained with 2% SYTO RNA Select green fluorescent cell stain and used as control. To remove the remaining dye, samples were centrifuged on exosome spin columns (MW 3000, Thermo Fisher Scientific) according to manufacturer's instructions. Exosomes concentration was indirectly measured in a Bradford assay. Finally, after IVF, 25 murine zygotes were co-cultured with labeled EV-endMSCs at different concentrations or stained PBS as negative control. Internalization (green fluorescence) was confirmed by fluorescent microscopy. Additionally, embryos were stained with 2.5 mg/ml of Hoechst 33342 (Eugene, OR, USA) for 10 min at 37°C to visualize DNA.
10
Biodistribution of Cell-Derived Extracellular Vesicles in Mice
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To assess the biodistribution of the EVs in mice, we used 5 µl/ml DiD (KGMP0025, KeyGEN BioTECH, China)-labeled HK-2 cell-derived EVs, and the unbound dye was removed with exosome spin columns (MW3000, Invitrogen). Then, 100 µg of HK-2 cell-derived EVs was injected into C57BL/6J mice (8 weeks old) via the caudal vein. After 24 h, the tissue samples were harvested and observed using an in vivo imaging system (IVIS) spectrum (Ami HTX, Spectral Instruments Imaging, USA).
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