ReN cells were maintained as described by Kim, Y.H. et all [32 (link)]. Briefly, ReN cells (Millipore) were maintained in Proliferation medium (484.5 ml DMEM/F12 (Gibco/Life Technologies) with 0.5 ml of heparin (2 mg/ml stock, STEMCELL Technologies), 10 ml of B27 (Life Technologies) 5 ml of 100X penicillin/streptomycin/amphotericin B (Lonza), 80 μl of bFGF stock and 100 μl of EGF stock) on Matrigel (Sigma-Aldrich) coated flasks at 37°C CO2 incubator. For differentiation the media were changed to Differentiation media, which is Proliferation media containing no growth factors, bFGF or EGF. The cells were maintained in Differentiation media for ~ 6 days to obtain neuronal structure prior to co-IP assays.
Ren cells
Manufactured by Merck Group
Sourced in United States
ReN cells are a type of neural progenitor cells derived from human embryonic stem cells. They exhibit properties of neural precursor cells and have the potential to differentiate into various neural cell types. The core function of ReN cells is to serve as a model system for studying neural development, neurological diseases, and drug screening applications.
Automatically generated - may contain errors
Lab products found in correlation
Dmem f12, by Thermo Fisher Scientific (5 mentions)
Basic fibroblast growth factor (bfgf), by Applied Biological Materials (5 mentions)
Epidermal growth factor (egf), by Applied Biological Materials (5 mentions)
Mini extruder, by Avanti Polar Lipids (3 mentions)
Polycarbonate porous membrane, by Cytiva (3 mentions)
Accutase, by Thermo Fisher Scientific (3 mentions)
Zetaview system, by Particle Metrix (3 mentions)
Tecnai g2 transmission electron microscope, by Thermo Fisher Scientific (3 mentions)
Whatman, by Cytiva (3 mentions)
Matrigel, by Merck Group (1 mentions)
Heparin, by STEMCELL (1 mentions)
Penicillin streptomycin amphotericin b, by Lonza (1 mentions)
Dmem f12 medium, by Thermo Fisher Scientific (1 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
Hek293t cells, by Thermo Fisher Scientific (1 mentions)
6 protocols using ren cells
1
Differentiation of ReN Neural Progenitor Cells
2
Labeling of extracellular vesicles from neural progenitor cells
3
Labeling Neural Stem Cells and Extracellular Vesicles
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ReN cells were purchased from Millipore (Billerica, USA). The cells were cultured in DMEM/F12 (Life Technologies, Grand Island, USA) supplemented with 2% B27 (Life Technologies), 20 μg/mL EGF (Abm, Richmond, Canada), 10 μg/mL bFGF (Abm) in an incubator with 5% CO2 at 37 °C. BV2 and human embryonic kidney (HEK293T) cells (Type Culture Collection of the Chinese Academy of Sciences, Shanghai, China) were maintained in DMEM (Life Technologies) containing 10% fetal bovine serum (FBS) (PAN, Aidenbach, Germany) in a CO2 incubator. For HEK293T cell-derived EVs (EV293) isolation, EV-depleted FBS (prepared by overnight centrifugation at 200,000 g at 4 °C) was used. To label EVs with tdTomato, ReN cells were stably transduced with packaged lentivirus vectors to express tdTomato fused to palmitoylation signal (palm-tdTomato) which labels cell membrane 30 (link). In addition, to display Gaussia luciferase (Gluc) on EV surface, lentivirus expressing Gluc fused to the transmembrane domain (TM) of the platelet-derived growth factor receptor (Gluc-TM) was packaged to stably transduce ReN cells 11 (link). Both of Palm-tdTomato and Gluc-TM constructs have been verified in previous studies 11 (link),30 (link).
4
Isolation and Characterization of ReN-NV
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ReN cells were purchased from Millipore (USA). The cells with passages of 5–10 were cultured in DMEM/F12 (Life Technologies, USA) supplemented with 2% B27 (Life Technologies), 20 ng/mL EGF (Abm, Canada), 10 ng/mL bFGF (Abm) and 2 μg/mL heparin and incubated with 5% CO2 at 37 °C. ReN cell spheres were dissociated with Accutase (Life Technologies). The cells were centrifuged at 3000×g for 10 min to remove the nucleus after homogenate by ultrasonication. The supernatants were extruded serially through 1 μm, 400 nm and 200 nm polycarbonate porous membranes (Whatman, UK) using a mini extruder (Avanti Polar Lipids, USA). Subsequently, the extruded ReN-NV were purified in a layer between 10% and 50% (v/v) iodoxanol by ultracentrifugation (L-80XP, Bechman Coulter, USA) at 100,000×g for 2 h. The ReN-NV pellet was resuspended in PBS after a wash step by another ultracentrifugation with PBS.
ReN-NV were observed by a Tecnai G2 transmission electron microscope (FEI, USA). The total protein concentration was determined by BCA protein assay (Pierce, USA). The size distribution and number were analyzed by NTA using a ZetaView system (Particle Metrix, Germany). As a control, HEK293 cell-derived NV (293-NV) were prepared and identified following an identical procedure.
ReN-NV were observed by a Tecnai G2 transmission electron microscope (FEI, USA). The total protein concentration was determined by BCA protein assay (Pierce, USA). The size distribution and number were analyzed by NTA using a ZetaView system (Particle Metrix, Germany). As a control, HEK293 cell-derived NV (293-NV) were prepared and identified following an identical procedure.
5
Isolation and Characterization of ReN-Derived Nanovesicles
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ReN cells were purchased from Millipore (USA). The cells with passages of 5-10 were cultured in DMEM/F12 (Life Technologies, USA) supplemented with 2% B27 (Life Technologies), 20 μg/mL EGF (Abm, Canada), 10 μg/mL bFGF (Abm) and 2 μg/mL heparin and incubated with 5% CO 2 at 37 °C. ReN cell spheres were dissociated with Accutase (Life Technologies). The cells were centrifuged at 3,000 × g for 10 min to remove the nucleus after homogenate by ultrasonication. The supernatants were extrudedserially through 1 μm, 400 nm and 200 nm polycarbonate porous membranes (Whatman, UK) using a mini extruder (Avanti Polar Lipids, USA). Subsequently, the extruded ReN-NV were puri ed in a layer between 10% and 50% (v/v) iodoxanol by ultracentrifugation (L-80XP, Bechman Coulter, USA) at 100,000 × g for 2 h. The ReN-NV pellet was resuspended in PBS after a wash step by another ultracentrifugation with PBS.
ReN-NV were observed by a Tecnai G2 transmission electron microscope (FEI, USA). The total protein concentration was determined by BCA protein assay (Pierce, USA). The size distribution and number were analyzed by NTA using a ZetaView system (Particle Metrix, Germany). As a control, HEK293 cell-derived NV (293-NV) were prepared and identi ed following an identical procedure.
ReN-NV were observed by a Tecnai G2 transmission electron microscope (FEI, USA). The total protein concentration was determined by BCA protein assay (Pierce, USA). The size distribution and number were analyzed by NTA using a ZetaView system (Particle Metrix, Germany). As a control, HEK293 cell-derived NV (293-NV) were prepared and identi ed following an identical procedure.
6
Extracellular Vesicle Isolation from ReN Cells
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ReN cells were purchased from Millipore (USA). The cells were cultured in DMEM/F12 (Life Technologies, USA) supplemented with 2% B27 (Life Technologies), 20 μg/mL EGF (Abm, Canada), 10 μg/mL bFGF (Abm) and 2 μg/mL heparin and incubated with 5% CO 2 at 37 °C. ReN cell spheres were dissociated with Accutase (Life Technologies). The cells were centrifuged at 3,000 × g for 10 min to remove the nucleus after homogenate by ultrasonication. The supernatants were extruded serially through 1 μm, 400 nm and 200 nm polycarbonate porous membranes (Whatman, UK) using a mini extruder (Avanti Polar Lipids, USA). Subsequently, the extruded ReN-NV were puri ed in a layer between 10% and 50% (v/v) iodoxanol by ultracentrifugation (L-80XP, Bechman Coulter, USA) at 100,000 × g for 2 h. The ReN-NV pellet was resuspended in PBS after a wash step by another ultracentrifugation with PBS.
ReN-NV were observed by a Tecnai G2 transmission electron microscope (FEI, USA). The total protein concentration was determined by BCA protein assay (Pierce, USA). The size distribution and number were analyzed by NTA using a ZetaView system (Particle Metrix, Germany). As a control, HEK293 cell-derived NV (293-NV) were prepared and identi ed following an identical procedure.
ReN-NV were observed by a Tecnai G2 transmission electron microscope (FEI, USA). The total protein concentration was determined by BCA protein assay (Pierce, USA). The size distribution and number were analyzed by NTA using a ZetaView system (Particle Metrix, Germany). As a control, HEK293 cell-derived NV (293-NV) were prepared and identi ed following an identical procedure.
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