One mL of human plasma was incubated with 0.5 mL of Invitrogen total exosome isolation reagent (Thermo Fisher Scientific) overnight at 4°C, then centrifuged at 10,000 x g for 1h at 4°C. Pellets were resuspended in 2 volumes of radioimmunoprecipitation assay buffer (RIPA, 150 μL, 50 mM Tris-HCI pH 7.4, 1% NP40, 0.5% Na-deoxycholate, 0.1% SDS, 150 mM NaCI, 2mM EDTA), homogenized and sonicated. Thirty μg of protein was used for western blot analyses. Cell media from 6-OHDA- or vehicle-treated SH-SY5Y cells were collected after 8 h of incubation and centrifuged at 800 x g for 10 min at 4°C. Supernatants were filtered using a 0.22 μm filter and centrifuged at 100,000 x g for 90 min at 4°C. Pellets were resuspended in 0.5 mL PBS containing a proteinase inhibitor cocktail (Thermo Fisher Scientific) and centrifuged again at 100,000 x g for 90 min at 4°C. Supernatants were collected and 30 μg of protein were used for western blot analyses to verify exosome markers (CD63 and Alix).
Invitrogen total exosome isolation reagent
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Invitrogen Total Exosome Isolation Reagent is a product designed for the isolation of exosomes from various sample types, including cell culture media and biological fluids. It utilizes a polymer-based precipitation method to effectively separate exosomes from the surrounding biological matrix.
Automatically generated - may contain errors
Lab products found in correlation
Proteinase inhibitor cocktail, by Thermo Fisher Scientific (1 mentions)
Ns300, by Malvern Panalytical (1 mentions)
Nunc lab tek 2 chambered coverglasses, by Thermo Fisher Scientific (1 mentions)
Invitrogen total exosome isolation kit, by Thermo Fisher Scientific (1 mentions)
A2720803, by Thermo Fisher Scientific (1 mentions)
6 protocols using invitrogen total exosome isolation reagent
1
Isolation and Characterization of Extracellular Vesicles from SH-SY5Y Cells
2
Exosome Isolation and Characterization using NTA
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Cell supernatant from MCF7 cells in 90 mm dishes was used to isolate exosome pellet using exosome isolation reagent (Invitrogen Total Exosome Isolation Reagent, Thermo Fisher) following the manufacturer’s protocol and diluted up to 1 ml with exosome-free 1X PBS. Using a syringe pump, 1 ml of each sample was injected into the field under UV in the nanoparticle tracking system (Nanosight, NS300, Malvern). 5 videos at 1 min each per sample were acquired with camera level 16, detection threshold 5 or 6 at temperature 25 °C and maximum jump length, blur, minimum track length set to auto.
3
Exosome Isolation and SGN Culture
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Exosomes were purified from the conditioned media using Invitrogen Total Exosome Isolation Reagent (from cell culture media; Cat#: 4478359, ThermoFisher) according to the manufacturer’s instructions. The explants or SGNs were cultured in 400 μl SGN basic medium in Nunc Lab-Tek II Chambered Coverglasses (Cat#: 155409, ThermoFisher) with exosomes (5 μg/ml), with or without SU5408 (100 nM for SGN explant and 50 nM for SGNs) for five continuous days. Media was changed every 2 d.
4
Exosome Isolation from Serum
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Serum samples were centrifuged by 2,000 × g for 30 min at 4°C to remove cell debris. Supernatants were transferred to new microcentrifuge tubes and mixed with Invitrogen
Total Exosome Isolation reagent (Thermo Fisher Scientific). After incubation at 2–8°C for 30 min, centrifugation was conducted by 10,000 × g for 10 min at room temperature.
The pellet was collected and re-suspended with cold phosphate buffer saline (PBS) filtered with the 0.22 μm pore-size membrane. Purified EVs were measured for the size and concentration
using a nanoparticle imaging analyzer (Videodrop, Myriade, France).
Total Exosome Isolation reagent (Thermo Fisher Scientific). After incubation at 2–8°C for 30 min, centrifugation was conducted by 10,000 × g for 10 min at room temperature.
The pellet was collected and re-suspended with cold phosphate buffer saline (PBS) filtered with the 0.22 μm pore-size membrane. Purified EVs were measured for the size and concentration
using a nanoparticle imaging analyzer (Videodrop, Myriade, France).
5
Exosome Isolation from Murine Samples
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At the end of the studies, mice were euthanized with isoflurane and CO2 inhalation and blood samples were withdrawn from the left femoral artery. Exosomes were isolated from blood samples using Invitrogen Total Exosome Isolation Kit (from plasma) (Invitrogen 4484450, Thermo Fisher Scientific, Waltham, MA, USA). For the isolation of exosomes from cell cultures, Invitrogen Total Exosome Isolation Reagent (from cell culture media) (Invitrogen 4478359, Thermo Fisher Scientific, Waltham, MA, USA) was used. All of the reagents were provided by the kits, and procedures adhered to the manufacturers’ instructions. The use of exosome isolation kits and protocols was referred to relevant studies [21 (link),22 (link)].
6
Dengue Virus Infection and Extracellular Vesicle Isolation in Aedes aegypti Cells
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Aedes aegypti (ATC-10) (ATCC® CCL-125™, Manassas, VA, USA) cells were cultured in DMEM (Gibco™ 11995065) containing 10% FBS (Gemini 100–106), 1% penicillin/streptomycin (Gibco™ 15140163), and 1% tryptose phosphate broth (Gibco™ 18050039) at 30 °C with 5% CO2. The cells were treated with Dengue Virus type 2, New Guinea Strain (DENV-2-NGC) at MOI of 1.0. Following 2 h of treatment with DENV-2, the media and virus were aspirated, and the cells were washed three times with sterile PBS (Gibco™ 10010049) and cultured in DMEM containing 10% exosome-depleted FBS (Gibco™ A2720803, Thermo Scientific, Waltham, MA, USA), 1% penicillin/streptomycin (Gibco™ 15140163), and 1% tryptose phosphate broth (Gibco™ 18050039, Thermo Scientific, Waltham, MA, USA) for 72 h. The cell supernatants following infection or not were collected and used to isolate extracellular vesicles using Invitrogen™ Total Exosome Isolation Reagent (Invitrogen™ 4478359, Thermo Scientific, Waltham, MA, USA), a method previously described for the isolation of EVs from Aedes cells [27 (link),55 (link)], according to the manufacturer’s instructions. The proteins contained in these EVs were processed using the Thermo Scientific Pierce In-Solution Tryptic Digestion and Guanidination Kit (Thermo Scientific™ 89895, Waltham, MA, USA) according to the manufacturer’s instructions.
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