The fluorescent reagent Exo-Green (System Biosciences) was used to label the protein component of huMSC-EXOs, and Exo-Red (System Biosciences) was used to label the microRNAs in huMSC-EXOs. HuMSC-EXOs were labelled with Exo-Green and Exo-Red, separately, for 20 min at 37 °C, and then, the labelled huMSC-EXOs were washed with PBS and re-pelleted twice using ExoQuick-TC exosome precipitation solution (System Biosciences). Exo-Green-labelled huMSC-EXOs (100 µg, 100 µg/ml) were incubated with OGCs in a six-well plate for 2 h at 37 °C, whilst Exo-Red-labelled huMSC-EXOs were incubated for 24 h. Images were acquired with a fluorescence microscope.
Exo red
Manufactured by System Biosciences
Sourced in United States
Exo-Red is a fluorescent labeling kit designed for the detection of extracellular vesicles (EVs), such as exosomes, in cell culture media and biological fluids. The kit provides a simple and efficient method for labeling EVs with a red fluorescent dye, allowing for their visualization and analysis using fluorescence-based techniques.
Automatically generated - may contain errors
Lab products found in correlation
Exo green, by System Biosciences (15 mentions)
Exoquick tc, by System Biosciences (9 mentions)
Differentiated human adipocytes, by Lonza (6 mentions)
Sp5 tandem scanner spectral 2 photon confocal microscope, by Leica (6 mentions)
Murine 3t3 l1 cells, by American Type Culture Collection (6 mentions)
Pkh67, by Merck Group (6 mentions)
Exoquick tc exosome precipitation solution, by System Biosciences (6 mentions)
Fv100, by Olympus (3 mentions)
8 well chamber slide, by BD (3 mentions)
Facsaria 2, by BD (3 mentions)
Evos fl auto 2, by Thermo Fisher Scientific (3 mentions)
Lsm 510 meta nlo confocal microscope, by Zeiss (3 mentions)
Facscalibur, by BD (3 mentions)
Pkh67 green, by Merck Group (3 mentions)
Exo flow ip kit, by System Biosciences (3 mentions)
9 protocols using exo red
1
Labeling and Tracking Exosomes
2
Exosome Labeling and Uptake Assay
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Exosomes were labeled with EXO-Red (System Biosciences, Mountain View, CA) according to the manufacturer's recommendations. MIA PaCa-2, BxPC-3 cells and THP-1 cells were plated at 2.5×104 cells/well in 8 well chamber slides (BD Falcon, Bedford, MA), allowed to adhere for 24 h in serum-containing medium, washed 3x with PBS, cultured for 2 h at 37°C in serum-containing medium supplemented with 20 µg/mL EXO-Red-labeled PANC-1 exosomes or 0.5 µg/mL recombinant fluorescent-labeled EphA2 protein. THP-1 monocytes were treated with 50 ng/mL phorbol 12-myristate 13-acetate (PMA) to differentiate into macrophage. Recombinant EphA2 was labeled with Alexa Fluor 488 dyes according to the manufacturer's instructions. For microscopy studies, cells were washed 3x with PBS, incubated for 15 min at 25°C with 4% paraformaldehyde, and incubated for 5 min at 25°C in 1:1000 4'6-diamidino-2-phenylindole (DAPI)/PBS solution, and exosomes were visualized using a laser scanning confocal microscope with a 40× objective (FV-100; Olympus). For flow cytometry studies, cells were PBS washed, detached, PBS washed, diluted in PBS with 10% BSA, and then analyzed on a BD FACS AriaII (BD Biosciences, San Jose, CA) to measure exosome uptake by florescence intensity 44 (link).
3
Exosome Labeling and Uptake Assay
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Purified exosomes from either human or mouse plasma samples were labeled with green fluorescent linker PKH67 (Sigma-Aldrich, St. Louis, MO), Exo-Red, and Exo-Green (System Biosciences, Mountain View, CA), and further incubated at 37°C for 10 min, until the reaction was stopped by adding ExoQuick-TC reagent. Labeled exosomes were added to either differentiated human adipocytes (# PT-5006, Lonza, Walkersville, MD) or murine 3T3-L1 cells (ATCC, Manassas, VA) for 4 hours in a cell culture incubator at 37°C. Labeled exosomes were monitored for delivery into target cells using a Leica SP5 Tandem Scanner Spectral 2-photon confocal microscope (Leica Microsystems, Inc., Buffalo Grove, IL) with a 63× oil-immersion lens.
4
Exosome Labeling and Uptake Assay
5
Exosome Uptake Quantification in Target Cells
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Exosomes were isolated from conditioned media of CDCs and resuspended in 500 ul of 1x PBS. Exosomes were labeled with Exo-Red (Acridine Orange chemistry) (System Biosciences) as previously described7 (link). Target cells were incubated with labeled exosomes and their uptake was assessed by fluorescence microscopy (Invitrogen EVOS FL Auto 2) at 2 and 24 hours at both 10x and 20x magnification. Parameters were controlled between image acquisition steps by creating an automatic scanning protocol. Fluorescent-labeled exosome uptake was quantified with ImageJ using the Color Threshold technique adapted from Jensen30 (link) with slight modifications. Briefly, a color threshold was applied to each image with the following parameters: Hue: 0–255, Saturation: 0–255, Brightness: 31–255, Threshold method: Triangle. After thresholding, the Integrated Density (pixel area × mean gray value) was calculated for each image. Images were adjusted and scaled identically, and no scaling units were added post-image acquisition.
6
Exosome Labeling and Uptake Assay
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Exosomes were isolated from conditioned media of cells at P4 as described above and resuspended in 500ul of 1x PBS. 50ul of 10x Exo-Red (Acridine Orange chemistry) or Exo-Green (Carboxyfluorescein succinimidyl diacetate ester (CFSE) chemistry) (System Biosciences) was added to 500ul of resuspended exosomes suspension, inverted to mix, and incubated at 37°C for 10 minutes. To stop the reaction, 100ul of ExoQuick-TC was added (System Biosciences) and the suspension was mixed by inverting 6 times. The labeled exosome sample was placed on ice for 30 minutes then centrifuged for 3 minutes at 14,000rpm. The supernatant was removed and the labeled exosome pellet was resuspended in 500ul of 1x PBS. Target cells were incubated with labeled exosomes and their uptake assessed by flow cytometry (BD FACSCalibur) and confocal microscopy (Zeiss LSM 510 Meta NLO Confocal Microscope) at various time points.
7
Labeling and Tracking Extracellular Vesicles
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Purified exosomes were labeled with fluorescent linker Exo-Red (System Biosciences, Mountain View, CA), Exo-Green (System Biosciences, Mountain View, CA, USA), or PKH67-Green (Sigma-Aldrich, St. Louis, MO, USA), and further incubated at 37 °C for 10 min. To remove unbound dyes, samples were filtered through a microspin column G-25 (Sigma-Aldrich, St. Louis, MO, USA). The reactions of all samples were stopped by adding ExoQuick-TC reagent, followed by placing the labeled exosome samples at 4 °C for 40 min and centrifugation for 3 min at 14,500× g. The pellets were suspended in 1× PBS, and the labeled exosomes were added to human adipocytes, hepatocytes, and myocytes for 6 h in a cell culture incubator at 37 °C. See Supplementary Methods for further details.
8
Exosome Labeling and Isolation Protocol
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Isolated exosomes from serum or cell supernatants were suspended in 1 ml of 1X PBS. Exosome aliquots (500 μl) were labeled with 50 μl of 10X Exo-Red [SBI] according to manufacturer’s instructions. The exosomes were re-isolated using the addition of 100 μl ExoQuick followed by precipitation for 30 min at +4 °C. The labeled exosome pellet was suspended in 500 μl 1X PBS and stained with CD63-coupled magnetic beads provided by SBI’s Exo-Flow IP kit [SBI] and with the FITC-conjugated peptides.
9
Visualization of EV Uptake in TM Cells
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Cellular uptake of the NPCE RNA and proteins via EVs was analysed using image stream. TM cells (5 × 105) were seeded in a 6‐well plate in high glucose DMEM, containing 10% EV‐depleted FBS. After 24 hours, the TM cells were incubated for 2 hours with Exo‐Red (EXOC300A‐1, SBI, CA, USA) or Exo‐Green (EXOC300A‐1, SBI) stained EVs isolated from NPCE cells. Labelling of exosomal RNA by Exo‐Red and proteins by Exo‐Green was performed according to the manufacturer's protocol. 10 μL of Exo‐Red or Exo‐Green was added to 20 μg of EVs resuspended in 500 μL 1×PBS. The EV suspension was mixed with the stain solution and incubated for 10 minutes at 37°C. The labelling reaction was stopped by adding ExoQuick‐TC reagent. Labelled EVs were placed on ice for 30 minutes following centrifugation at 17136 g for 3 minutes. 100 μL of labelled NPCE EVs were co‐cultured with TM cells for two hours washed and analysed on the ImageStreamX Mark II imaging cytometer equipped with 60× objective. Exo‐Green and Exo‐Red fluorescence were recorded using excitation with 488 nm and 642 nm lasers, respectively at 3 mW intensity.
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