Liposomes and EVs were labeled with BODIPY FL C5-HPC membrane dye as described above. EVs enriched pellet were resuspended in 10 μL of TAE (TRIS 39 mM, acetic acid glacial 19,9 mM, EDTA 1,27 mM) plus 0.02% SDS and then loaded on a 0.6% agarose gel and electrophoresed 30 min at 100 V. Fluorescent signal was acquired using a G:Box Chemi XT Imaging system (Syngene). Then the gel was stained with Coomassie Brilliant Blue for 30 minutes at room temperature and destained in 10% acetic acid, 20% methanol overnight. Images were acquired using a G:Box Chemi XT Imaging system (Syngene).
G box chemi xt imaging system
Manufactured by Syngene
The G:Box Chemi XT Imaging system is a versatile laboratory equipment designed for the detection and analysis of chemiluminescent, fluorescent, and colorimetric signals. It is capable of capturing high-quality images of a wide range of samples, including blots, gels, and microplates.
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4 protocols using g box chemi xt imaging system
1
EV Labeling and Gel Electrophoresis
2
Liposome and Exosome Fluorescent Imaging
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Liposomes, P3, Exo PK and fractions from different gradients were labeled with PKH67 membrane dye as described above. Samples were loaded on a 0.6% agarose gel and electrophoresed 30 min at 100 V as previously reported28 (link). Fluorescent signal was acquired using a G:Box Chemi XT Imaging system (Syngene). The gel was, then, stained with Coomassie Brilliant Blue for 1 h at 37 °C and destained with Destaining solution (10% Acetic acid, 20% Methanol). Images were acquired using a G:Box Chemi XT Imaging system (Syngene).
3
Quantifying Extracellular Vesicle Protein Content
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Extracellular vesicles protein concentration from controls (three patients), MGUS (three patients), and MM (six patients) serum was quantified with Bradford assay. Samples were normalized for protein content (200 μg). EVs were PKH67-labeled as described and re-suspended in 20 μl of loading dye [36% Tris–acetate–EDTA (TAE), 14% H2O, 50% glycerol]. Ten microliters of fluorescent EVs were spotted on a nitrocellulose membrane and dots fluorescent signal were acquired using a G:Box Chemi XT Imaging system (Syngene). Signals were quantified with the Gene Tools program. Protein content/lipid fluorescent signal ratio was calculated.
4
Western Blot Analysis of EV Proteins
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Equivalent amounts of cells and EV protein lysates were electrophoresed and transferred to nitrocellulose membranes. Nitrocellulose membranes were then blocked in 5% non-fat milk or 5% BSA, 10 mmol/l Tris-HCl pH 7.5, 100 mmol/l NaCl, 0.1% Tween-20, and probed with the following primary antibodies: mouse monoclonal anti-GM130 (1:1000, BD Biosciences, 610822), mouse monoclonal anti-Annexin V (1:500, Santa Cruz Biotechnology, Dallas, U.S., sc-74438), mouse monoclonal anti-Annexin XI (1:500, Genetex, CA, U.S., GTX33010), mouse monoclonal anti-TSG101 (1:500, Santa Cruz Biotechnology, sc-7964) rabbit monoclonal anti-TERT (1:500 1000 Rockland Immunochemicals Inc., PA, U.S., 600-401-252S), mouse monoclonal anti-Pro-COL1A2 (D-6) (1:200, Santa Cruz Biotechnology, sc-166572), rabbit monoclonal anti-Integrin beta1 (1:2000, ab179471), rabbit monoclonal anti-MMP3 (1:1000, ab52915), and rabbit monoclonal anti-MMP14 (1:2000, ab51074) (all purchased by Abcam) and incubated in the presence of specific, HRP-conjugated secondary antibodies. Immunoreactive bands were visualized using ECL detection system (Amersham International, L.C., UK) and by ChemiDoc XRS Imaging system (BioRad, CA, U.S.) or G:Box Chemi XT Imaging system (Syngene, Cambridge, UK).
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