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100 kda mwco

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The 100-kDa MWCO (Molecular Weight Cut-Off) is a laboratory equipment designed for membrane filtration processes. It is capable of separating and concentrating molecules, proteins, or other macromolecules based on their molecular weight. The core function of this product is to allow the passage of molecules smaller than 100 kDa while retaining larger molecules on the membrane surface.

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Lab products found in correlation

Hek293ft cells, by Thermo Fisher Scientific (3 mentions) Custom taqman assay, by Thermo Fisher Scientific (3 mentions) Benzonase, by Merck Group (2 mentions) 15 cm tissue culture dishes, by Corning (2 mentions) Universal nuclease, by Thermo Fisher Scientific (2 mentions) Taqman assay, by Thermo Fisher Scientific (1 mentions) Optima l 80 xp, by Beckman Coulter (1 mentions) 0.22 μm filter, by Merck Group (1 mentions) 0.22 μm pore filter, by Merck Group (1 mentions) Quickchange 2 site directed mutagenesis kit, by Agilent Technologies (1 mentions)

Spelling variants of this product

Amicon Ultra 100 kDa MWCO (8 citations) Amicon Ultra 100 kDa MWCO filters (6 citations) Amicon Ultra-4 Centrifugal Filter 100 kDa MWCO (3 citations) Amicon-Ultra 100 kDa MWCO spin filter units (2 citations) 100 kDa MWCO concentrators (2 citations) Amicon Ultra-15 Centrifugal Filter 100 kDa MWCO (2 citations) Amicon Ultra-15 100 kDa MWCO centrifugal concentrator (2 citations) 100 kDa MWCO spin concentrator (2 citations) Amicon 100 kDa MWCO Ultra Centrifugal filters (2 citations)

8 protocols using 100 kda mwco

1

Efficient AAV Purification Protocol

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Various AAV knockout plasmids used in this study as described in the figures and results were subjected to AAV production and chemical purification. Briefly, HEK293FT cells (ThermoFisher) were transiently transfected with transfer (AAV-plasmids), serotype (AAV9 or AAV6) and packaging (pDF6) plasmids using polyethyleneimine (PEI). Approximately 72 h post-transfection, cells were dislodged and transferred to a conical tube in sterile PBS. 1/10 volume of pure chloroform was added to the mixture and incubated at 37°C for 1 h. NaCl was added to a final concentration of 1 M. The mixture was subsequently shaken until dissolved and then pelleted at 20,000gx at 4°C for 15 min. The chloroform layer was discarded while the aqueous layer was transferred to another tube. PEG8000 was added to 10% (w/v) and shaken until dissolved. The mixture was incubated at 4°C for 1 h and spun at 20,000gx at 4 °C for 15 min. After the supernatant was discarded, the pellet was resuspended in DPBS + MgCl2, treated with benzonase (Sigma), and then incubated at 37°C for 30 min. Chloroform (1:1 volume) was then added, shaken and spun down at 12,000g at 4°C for 15 min. The aqueous layer was isolated and passed through a 100-kDa MWCO (Millipore). The concentrated solution was washed with PBS and the filtration process was repeated. Virus was titered by qPCR using custom Taqman assays (ThermoFisher).
Dong M.B., Wang G., Chow R.D., Ye L., Zhu L., Dai X., Park J.J., Kim H.R., Errami Y., Guzman C.D., Zhou X., Chen K., Renauer P.A., Du Y., Shen J., Lam S., Zhou J., Lannin D.R., Herbst R.S, & Chen S. (2019). Systematic immunotherapy target discovery using genome-scale in vivo CRISPR screens in CD8 T cells. Cell, 178(5), 1189-1204.e23.
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2

Exosome Labeling and In Vivo Tracking

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According to the manufacturer's instructions, exosomes were incubated with the cross-linkable membrane dye, CM-DiR (Ruitai bio, Beijing, China), for 30 min at 37°C in the dark. After washing with PBS, the labeled exosomes were concentrated with a 100 kDa MWCO (Millipore, Billerica, Massachusetts, USA) at 1000 × g for 30 min at 4°C to remove nonbinding dye. Then CM-DiR-labeled exosomes were injected into CCl4-induced mice via the tail vein. After injection for 24 h, mice were imaged using a Maestro in Vivo Imaging System (CRI) to observe the distribution of exosomes.
Jiang W., Tan Y., Cai M., Zhao T., Mao F., Zhang X., Xu W., Yan Z., Qian H, & Yan Y. (2018). Human Umbilical Cord MSC-Derived Exosomes Suppress the Development of CCl4-Induced Liver Injury through Antioxidant Effect. Stem Cells International, 2018, 6079642.
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3

Chloroform-based AAV6 and AAV9 Purification

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AAV6 and AAV9 were packaged and produced in HEK293FT cells and chemically purified by chloroform. In brief, HEK293FT cells were transiently transfected with the vector of interest, AAV serotype plasmid (AAV6 or AAV9), and pDF6 using polyethyleneimine (PEI). At 72 hr posttransfection, cells were dislodged and transferred to a conical tube in sterile DPBS. 1/10 volume of pure chloroform was added and the mixture was incubated at 37°C and vigorously shaken for 1 hr. NaCl was added to a final concentration of 1 M and the mixture was shaken until dissolved and then pelleted at 20,000 3 g at 4°C for 15 min. The aqueous layer was discarded while the chloroform layer was transferred to another tube. PEG8000 was added to 10% (w/v) and shaken until dissolved. The mixture was incubated at 4°C for 1 hr and then spun at 20,000 × g at 4°C for 15 min. The supernatant was discarded and the pellet was resuspended in DPBS plus MgCl2 and treated with Benzonase (Sigma) and incubated at 37°C for 30 min. Chloroform (1:1 volume) was then added, shaken, and spun down at 12,000 × g at 4C for 15 min. The aqueous layer was isolated and passed through a 100 kDa MWCO (Millipore). The concentrated solution was washed with DPBS and the filtration process was repeated. The virus was titered by qPCR using custom Taqman assays (Life Technologies) targeted to Cre.
Platt R.J., Chen S., Zhou Y., Yim M.J., Swiech L., Kempton H.R., Dahlman J.E., Parnas O., Eisenhaure T.M., Jovanovic M., Graham D.B., Jhunjhunwala S., Xavier R.J., Langer R., Anderson D.G., Hacohen N., Regev A., Feng G., Sharp P.A, & Zhang F. (2014). CRISPR-Cas9 Knockin Mice for Genome Editing and Cancer Modeling. Cell, 159(2), 440-455.
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4

AAV Production and Purification

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AAV was produced by transfecting HEK293FT cells (ThermoFisher) in 15-cm tissue culture dishes (Corning). Transfection was performed by using AAV2 transgene vectors, packaging (pDF6) plasmid, and AAV6/9 serotype plasmid together with polyethyleneimine (PEI). Transfected cells were collected using PBS after post-transfection 72 hours. For the AAV purification, transfected cells were mixed with pure chloroform (1/10 volume) and incubated at 37 °C with vigorously shaken for 1 h. NaCl was added to a final concentration of 1 M, and then the samples were centrifuged at 20,000g at 4 °C for 15 mins. The chloroform layer was discarded while the aqueous layer was transferred to another tube. PEG8000 was added to 10% (w/v) and shaken until dissolved. The mixture was incubated at 4 °C for 1 h and then centrifuged at 20,000g at 4 °C for 15 mins. The supernatant was discarded and the pellet was suspended in DPBS with MgCl2, treated with universal nuclease (ThermoFisher), and incubated at 37 °C for 30 mins. Chloroform (1:1 volume) was then added, shaken, and centrifuged at 12,000g at 4°C for 15 mins. The aqueous layer was isolated and concentrated through a 100-kDa MWCO (Millipore). Virus was titered by qPCR using custom Taqman assays (ThermoFisher) targeted to promoter U6.
Dai X., Park J.J., Du Y., Kim H.R., Wang G., Errami Y, & Chen S. (2019). One-step generation of modular CAR-T with AAV-Cpf1. Nature methods, 16(3), 247-254.
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5

Exosome Isolation from 2D and 3D Cell Cultures

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The 2D and 3D supernatants were centrifuged at 2000×g for 20 min and 13,500×g for 30 min at 4 °C to eliminate the cells and debris, followed by filtration with a 0.22-μm filter (Millipore) to remove microvesicles. Then, the supernatants were centrifuged at 200,000×g for 120 min (Type 70 Ti rotor, Beckman Coulter Optima L-80 XP) at 4 °C to deposit 2D-exos or 3D exosomes (3D-exos). The exosome pellets were resuspended in PBS and filtered using a 0.22-μm filter once again. The resuspended exosomes were concentrated using 100 KDa MWCO (Millipore) at 4000×g for 30 min. Finally, purified 2D-exos and 3D-exos were harvested in 200–400 μL of PBS and stored at − 80 °C.
Cao J., Wang B., Tang T., Lv L., Ding Z., Li Z., Hu R., Wei Q., Shen A., Fu Y, & Liu B. (2020). Three-dimensional culture of MSCs produces exosomes with improved yield and enhanced therapeutic efficacy for cisplatin-induced acute kidney injury. Stem Cell Research & Therapy, 11, 206.
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6

Isolation and Purification of Exosomes from hucMSC Conditioned Medium

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HucMSCs were isolated as previously described in our work [34 (link)]. All experiment protocols were approved by the Ethics Committee of Jiangsu University. The 70%–80% of the confluent hucMSCs cultures were washed twice with phosphate-buffered saline (PBS) and then incubated in serum-free low glucose Dulbecco's modified Eagle's medium (LG-DMEM) for 48 hours. The conditioned medium was collected and centrifuged at 1,000 ×g for 20 minutes to remove cell debris, followed by centrifugation at 2,000 ×g for 20 minutes and 10,000 ×g for 20 minutes. The supernatant was collected and concentrated using 100 KDa MWCO (Millipore, USA) at 1,000 ×g for 30 minutes. The concentrated supernatant was loaded upon 5 mL of 30% sucrose/D2O cushions and then ultracentrifuged at 100,000 ×g for 60 minutes (optimal-90K, Beckman Coulter). The microvesicles-enriched fraction was harvested and diluted with PBS and then centrifuged thrice at 1,000 ×g for 30 minutes using 100 KDa MWCO. Finally, the purified exosomes were collected and subjected to filtration on 0.22 μm pore filter (Millipore, USA) and stored at −70°C.
Zhang B., Shen L., Shi H., Pan Z., Wu L., Yan Y., Zhang X., Mao F., Qian H, & Xu W. (2016). Exosomes from Human Umbilical Cord Mesenchymal Stem Cells: Identification, Purification, and Biological Characteristics. Stem Cells International, 2016, 1929536.
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7

AAV Production and Purification

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AAV was produced by transfecting HEK293FT cells (ThermoFisher) in 15-cm tissue culture dishes (Corning). Transfection was performed by using AAV2 transgene vectors, packaging (pDF6) plasmid, and AAV6/9 serotype plasmid together with polyethyleneimine (PEI). Transfected cells were collected using PBS after post-transfection 72 hours. For the AAV purification, transfected cells were mixed with pure chloroform (1/10 volume) and incubated at 37 °C with vigorously shaken for 1 h. NaCl was added to a final concentration of 1 M, and then the samples were centrifuged at 20,000g at 4 °C for 15 mins. The chloroform layer was discarded while the aqueous layer was transferred to another tube. PEG8000 was added to 10% (w/v) and shaken until dissolved. The mixture was incubated at 4 °C for 1 h and then centrifuged at 20,000g at 4 °C for 15 mins. The supernatant was discarded and the pellet was suspended in DPBS with MgCl2, treated with universal nuclease (ThermoFisher), and incubated at 37 °C for 30 mins. Chloroform (1:1 volume) was then added, shaken, and centrifuged at 12,000g at 4°C for 15 mins. The aqueous layer was isolated and concentrated through a 100-kDa MWCO (Millipore). Virus was titered by qPCR using custom Taqman assays (ThermoFisher) targeted to promoter U6.
Dai X., Park J.J., Du Y., Kim H.R., Wang G., Errami Y, & Chen S. (2019). One-step generation of modular CAR-T with AAV-Cpf1. Nature methods, 16(3), 247-254.
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8

Producing N176Y Mutant Cx26 Protein

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Introduction of the N176Y mutation was performed using the human Cx26 C211S/C218S gene in the pVL1393 vector using the QuickChange II Site-Directed Mutagenesis Kit (Agilent). Protein expression and purification were performed as described for WT Cx26 (Bennett et al., 2016 (link)). Samples for EM were eluted from the IMAC column using a buffer containing 25 mM Tris pH 7.5, 500 mM NaCl, 2.5 % glycerol (v/v) and 0.025% façade-EM (FA-3; Anatrace, Maumee, OH). The final eluate from the SEC column was concentrated to 2–4 mg/ml using a 0.5 ml or 4 ml concentrator (100 kDa MWCO; Millipore). If protein was not used for experiments shortly after the purification, it was aliquoted into 0.6 ml tubes, flash-frozen in liquid nitrogen and stored at −80°C.
Khan A.K., Jagielnicki M., Bennett B.C., Purdy M.D, & Yeager M. (2021). Cryo-EM structure of an open conformation of a gap junction hemichannel in lipid bilayer nanodiscs. Structure (London, England : 1993), 29(9), 1040-1047.e3.
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