Conditioned medium was thawed and centrifuged at 300g for 5 min to pellet cell debris. The supernatant was transferred to fresh tubes and centrifuged at 2000g for 10 min to pellet larger extracellular particles. The supernatant was then passed through a 0.22-μm vacuum filter. Large volumes (up to 300 ml) of conditioned medium were concentrated through Sartorius Vivaspin 300-kDa molecular weight cutoff (MWCO) spin-filters at 2500g for 3 min to a volume of less than 20 ml. Filtrate was discarded, and retentate was removed and saved in a separate tube. The concentrated conditioned medium was then loaded onto a HiScreen Capto Core 700 column connected to an ÄKTA Pure 25 L Chromatography system (Cytiva Life Sciences). Flow rate settings for column equilibration, sample loading, and column clean in place procedure were chosen according to the manufacturer’s recommendations. The EV fraction was collected according to the 280-nm UV absorbance chromatogram and concentrated using Amicon Ultra-15 100-kDa MWCO spin-filters. Concentrated fractions were washed with 15-ml of phosphate-buffered saline (PBS) in the same spin-filter and concentrated to a final volume of 150 μl. All spins and chromatography were performed at 4°C. Samples were stored at 4°C for no more than 1 week before use in functional assays.
Hiscreen capto core 700 column
Manufactured by Cytiva
The HiScreen Capto Core 700 column is a laboratory equipment designed for chromatographic separation and purification of biomolecules. It features a stable and rigid core structure that provides high mechanical strength and uniform flow distribution. The column is compatible with a variety of aqueous buffers and can be used for a range of chromatographic techniques, such as ion exchange, hydrophobic interaction, and affinity chromatography.
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Lab products found in correlation
3 protocols using hiscreen capto core 700 column
1
Extracellular Vesicle Isolation Protocol
2
Isolation of Cell-Derived Extracellular Vesicles
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Cell-derived EVs were sequentially isolated by tangential flow filtration (TFF), bind-elute size exclusion chromatography (BE-SEC) and density gradient ultracentrifugation (DGUC). Briefly, HEK293F cells were cultured in CD05 medium for 96h, while MRC5 and HKB-11 cells were, respectively, cultured in fresh DMEM and DMEM-F12 medium without FBS for 48h before processing for EVs isolation. The supernatant of cultured cells was sequentially centrifuged at 800 ×g for 5min and 2000 ×g for another 10min to remove dead cells and debris and then filtered using a 0.2μm filter (Millipore, USA). To remove any free proteins, a hollow fiber TFF column (300kDa, MWCO) was used to treat the supernatant from the previous step, followed by purification with the BE-SEC column (HiScreen Capto Core 700 column, Cytiva) on an ÄKTA Pure 25 chromatography system (Cytiva). Finally, collected samples were purified by DGUC at 100,000 ×g for 18h at 4°C to obtain a continuous density gradient, as previously described.24 (link)
3
Exosome Purification from Breast Cancer
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A total of 4 ml PB was collected from each subject in an EDTA tube. Plasma was then isolated from each sample using Ficoll-Paque Plus Reagent (Cytiva) diluted with PBS at a ratio of 1:1, followed by centrifugation at 12,000 × g at 4°C for 15 min. Subsequently, bind-elute size exclusion chromatography columns (HiScreen Capto Core 700 column; Cytiva) connected to the ÄKTA Pure 25 chromatography system (Cytiva) were used to capture and purify exosomes from 1.5 ml plasma at room temperature. The columns were equilibrated with sterile PBS. The flow rate was 25 ml/min according to the manufacturer's instruction. Following exosome capture, reverse transcription-quantitative polymerase chain reaction (RT-qPCR) was carried out to assess the mRNA expression levels of ENAH and SEPT9 in the exosomes derived from the first batch of subjects (31 patients with breast cancer, 36 DCs and 14 HCs) and EGF, MMP-9 and CXCL8 in exosomes derived from the second batch of subjects (16 patients with breast cancer, 27 DCs and 19 HCs).
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