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Extraction and isolation of tissue exosomes min centrifuge tube cell membrane
Published by: ShannonJSutt (16) on Sun, Dec 4, 2022  |  Word Count: 860  |  Comments ( 0)  l  Rating
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Original Title: Extraction and Isolation of Tissue Exosomes Exosomes are released from intracellular multivesicular bodies after fusion with the cell membrane, which are about 30 ~ 150 nm in diameter Membranous vesicles. Almost all cells produce exosomes, which are secreted into various body fluids, The loop system reaches other cells and tissues and produces remote regulation. Traditional exosome-based functional mechanism studies are mainly based on certain cell supernatant-derived exosomes in vitro and in vivo. However, cell line cultures lack the complexity of the tumor microenvironment, in which there are interactions between cells; in vitro culture Trophoblasts may be altered in character and may not represent their tissue of origin well, whereas tissue exosomes may be more authentic More and more scholars begin to focus on the abundant information and communication of exosomes from various cells in the tissue microenvironment. Study on the functional mechanism of exosomes from coke to tissue. The extraction of tissue exosomes can refer to the following operation steps of Isolation and Extraction of Brain Tissue Exosomes. Related reagents: 1?Hibernate E Solution ?BrainBits, Springfield? 2, papain (Worthington) 3?Umibio Exosome Isolation Kit?Umibio, UR52121? 4?PBS Related equipment: 1. Homogenizer 2, Mesh filter 40-?m 3. Syringe filter 0.2 ?m 4. High-speed centrifuge 5, Vortex Oscillator Operation steps: I) Brain liquefaction treatment: 1. Fresh or previously frozen mouse hemispheres were dissected and added with Hibernate E solution (3 mL/hemisphere); 2, adding 20 units/mL of papain, and performing water bath at 37 deg C for 15 minutes; Expand the full text 3, adde that precooled Hibernate E with the same volume, and slightly homogenize the mixture on ice; 4. The brain homogenate was filtered through a 40-?m mesh filter; 5, filter that filtrate by a 0. 2-mu m filter, cbd centrifugal extractor ,cbd crystallization equipment, and collect the filtrate; (2) pretreatment of liquefy brain; 1. Centrifuge to remove debris: transfer the liquefied brain slurry to a centrifuge tube, and centrifuge at 3000 G at 4 ? for 10 min to remove debris in the filtrate; transfer the supernatant to a new centrifuge tube after centrifugation; 2, centrifuging to remove impurities: centrifuging the transferred supernatant at a 10000 of G at 4 deg C for 20 minutes to remove the impurities in the sample, and transferring the centrifuged supernatant into a new centrifuge tube; (3) extraction of brain exosomes; Exosomal particles were extracted by Umibio Exo some Isolation Kit. 1. Supernatant pretreatment: Exosomoe Concentration Solution (ECS reagent) is added to the centrifuged supernatant from which impurities are removed, and the specific dosage is as follows: Note: For other dosage specifications, please convert according to the reagent dosage in the table. 2. Solution mixing: After adding ECS reagent, cover the centrifuge tube tightly, mix it with a vortex oscillator for 1 min, and then place it at 2 ? to 8 ? for 2 H; 3. Precipitate exosomes: take out the centrifuge tube containing the mixed solution and centrifuge at 4 ? for 60 min at 10000 G, discard the supernatant, and the precipitate is rich in exosomes particles; (Note: try to absorb the supernatant as much as possible) 4. Resuspension of exosomes: Take 100 ?L of 1 × PBS to uniformly blow and centrifuge the precipitate, and transfer the resuspension to a new 1.5 mL centrifuge tube after it is uniformly suspended in PBS; 5. Harvest of exosomal particles: a 1.5 mL centrifuge tube containing the resuspension was centrifuged at 4 ° C for 2 min at 12000 G, and the supernatant, which was rich in exosomal particles, was retained.
(Note: If the centrifuged sediment is visible, centrifuge again.) 6, purifying the exosomes: transferring the obtained crude exosomes to the upper chamber of an Exosomoe Purafication Filter (EPF column), centrifuging at 3000 G for 10 minutes at 4 deg C, and collecting the liquid at the bottom of the EPF column tube after centrifugation, wherein the liquid is the purified exosomes; 7. Preservation of exosomes: Purified exosomes were preserved in a refrigerator at -80 ? for subsequent experiments. References: 1?J BiolChem. 2012 Dec 14; 287(51):43108-15. 2?J ExtracellVesicles. 2017 Jul 26; 6(1):1348885. From Gaobo Science Network Blog More information about exosomes can be obtained by adding WeChat 13146518829 and returning to Sohu to see more Responsible Editor: (function() { function getBrandHtml() { var brands = [],rotary vacuum evaporator, html = ''; for(var i = 0; i < brands.length; i++) { var brand = brands[i]; if(brands.length i+1) { html+= ''+brand.name+''; } else { html+= ''+brand.name+'?'; } } return html; }; if(document.getElementById('linkBtn')){ document.getElementById('linkBtn').onclick = function() { $('#brands').removeClass('brand');$ ( '# tipInfo').text ( 'Real name responded'); $ ('#linkBtn').remove();$ ('.real-response .content').css('line-height', '20px');$ ('.real-response .time').css('line-height', '20px'); }; document.getElementById('brands').innerHTML = getBrandHtml(); }; })();? toptiontech.com
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