hESC cultures were disaggregated using accutase for 20 minutes, washed using hESC media and pre-plated on gelatin for 1 hour at 37°C in the presence of ROCK inhibitor to remove MEFs. The nonadherent hESC were washed and plated on matrigel at a density of 10,000–25,000 cells/cm2 on matrigel (BD) coated dishes in MEF conditioned hESC media (CM) spiked with 10 ng/mL of FGF-2 and ROCK-inhibitor. Ideal cell density was found to be 18,000 cells/cm2. The ROCK inhibitor was withdrawn, and hESC were allowed to expand in CM for 3 days or until they were nearly confluent. The initial differentiation media conditions included knock out serum replacement (KSR) media with 10 nM TGF-b inhibitor (SB431542, Tocris) and 500 ng/mL of Noggin (R&D). Upon day 5 of differentiation, the TGF-b inhibitor was withdrawn and increasing amounts of N2 media (25%, 50%, 75%) was added to the KSR media every two days while maintaining 500 ng/mL of Noggin. For MS5 induction, established methods previously reported were used22 (link).
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Accutase
Accutase
Accutase is an enzymatic cell dissociation reagent used to detach adherent cells from culture surfaces.
It is a mixture of proteolytic and collagenolytic enzymes that gently dissociates cells while maintaining their viability and surface antigen expression.
Accutase is commonly used in stem cell research, cell culture, and regenerative medicine applications.
Its gentle dissociation properties make it a suitable alternative to trypsin for sensitive cell types.
PubCompare.ai's AI-driven platform can help researchers easily locate Accutase protocols from literature, pre-prints, and patents, and utilize comparison tools to identify the best products and protocols for their needs, streamlining the research process and optimizing results.
It is a mixture of proteolytic and collagenolytic enzymes that gently dissociates cells while maintaining their viability and surface antigen expression.
Accutase is commonly used in stem cell research, cell culture, and regenerative medicine applications.
Its gentle dissociation properties make it a suitable alternative to trypsin for sensitive cell types.
PubCompare.ai's AI-driven platform can help researchers easily locate Accutase protocols from literature, pre-prints, and patents, and utilize comparison tools to identify the best products and protocols for their needs, streamlining the research process and optimizing results.
Most cited protocols related to «Accutase»
4-(5-benzo(1,3)dioxol-5-yl-4-pyridin-2-yl-1H-imidazol-2-yl)benzamide
accutase
Cells
Culture Media, Conditioned
FGF10 protein, human
Fibroblast Growth Factor 2
Gelatins
Human Embryonic Stem Cells
Hyperostosis, Diffuse Idiopathic Skeletal
matrigel
noggin protein
Serum
Transforming Growth Factor beta
Images were acquired by collaborators from the Allen Institute for Cell Science, Seattle, as per Roberts and colleagues [34 (link)]. Briefly, wild-type C (WTC) hiPSCs were cultured in a feeder-free system on tissue culture dishes or plates coated with GFR Matrigel (Corning) diluted 1:30 in cold DMEM/F12 (Gibco). Undifferentiated cells were maintained with phenol red containing mTeSR1 media (85850, STEMCELL Technologies) supplemented with 1% (v/v) penicillin-streptomycin (P/S; Gibco). Cells were not allowed to reach confluency greater than 85% and are passaged every 3–4 days by dissociation into single-cell suspension using StemPro Accutase (Gibco). When in single-cell suspension, cells were counted using a Vi-CELL Series Cell Viability Analyzer (Beckman Coulter). After passaging, cells were replated in mTeSR1 supplemented with 1% P/S and 10 μM ROCK inhibitor (Stemolecule Y-27632, Stemgent) for 24 hours. Media is replenished with fresh mTeSR1 media supplemented with 1% P/S daily. Cells were maintained at 37°C and 5% CO2. Cells were maintained with phenol red–free mTeSR1 media (05876, STEMCELL Technologies) 1 day prior to live cell imaging.
Three to four days after cells are plated and mature and healthy colonies are observed on 96- and 24-well imaging plates, the cells are stained with NucBlue Live ready probe reagent (R37605, ThermoFisher) and CellMask Deep Red plasma membrane stain (C10046, ThermoFisher) to visualize DNA and plasma membrane, respectively. The protocol is available online:http://www.allencell.org/uploads/8/1/9/9/81996008/sop_for_cellmask-and-nucblue_v1.0_1.pdf . Phenol red–free mTeSR1 is preequilibrated to 37°C and 5% CO2. 1X NucBlue solution made in preequilibrated phenol red–free mTeSR1 is spun for 60 minutes at 20,000 g. The 2X and 10X working stocks of CellMask Deep Red lot #1730970 and #1813792, respectively, are made in 1X NucBlue solution. All solutions are kept at 37°C and 5% CO2 until used. The 100 μL and 400 μL of NucBlue solution are added per well of 96-well imaging plates and 24-well imaging plates, respectively, and incubated at 37°C and 5% CO2 for 20 minutes. An equal amount of CellMask Deep Red working stock is added to the wells containing NucBlue solution. Final dye concentrations in the wells are 1X NucBlue and 1X and 5X CellMask Deep Red lots #1730970 and #1813792, respectively. Cells are incubated at 37°C and 5% CO2 for 10 minutes and gently washed with preequilibrated phenol red–free mTeSR1. Fields of view as shown in Fig 4 that are acquired near the edge (and the center as a control) of hiPSC colonies receive an additional photoprotective cocktail treatment which serves to minimize singlet oxygen and free radical formation. The photoprotective cocktail is used at a working concentration of 0.3 U/ml (1:100) OxyFluor as defined by the OxyFluor product insert, with the addition of 10 mM sodium lactate and 1 mM ascorbic acid (OxyFluor OF-0005, Oxyrase).
As per Roberts and colleagues [34 (link)], cells were imaged on a Carl Zeiss spinning disk microscope with a Carl Zeiss 20×/0.8 NA plan APOCHROMAT or 100×/1.25 W C-APOCHROMAT Korr UV Vis IR objective, a CSU-X1 Yokogawa spinning disk head, and Hamamatsu Orca Flash 4.0 camera. Microscopes were outfitted with a humidified environmental chamber to maintain cells at 37°C with 5% CO2 during imaging. Cells are imaged immediately following the wash step and for up to 2.5 hours after dye addition on a Zeiss spinning disk microscope at 100× with the following general settings: 405 nm at 0.28 mW, 200 ms exposure; 638 nm at 2.4 mW, 200 ms exposure; acquiring each channel at each z-step.
Three to four days after cells are plated and mature and healthy colonies are observed on 96- and 24-well imaging plates, the cells are stained with NucBlue Live ready probe reagent (R37605, ThermoFisher) and CellMask Deep Red plasma membrane stain (C10046, ThermoFisher) to visualize DNA and plasma membrane, respectively. The protocol is available online:
As per Roberts and colleagues [34 (link)], cells were imaged on a Carl Zeiss spinning disk microscope with a Carl Zeiss 20×/0.8 NA plan APOCHROMAT or 100×/1.25 W C-APOCHROMAT Korr UV Vis IR objective, a CSU-X1 Yokogawa spinning disk head, and Hamamatsu Orca Flash 4.0 camera. Microscopes were outfitted with a humidified environmental chamber to maintain cells at 37°C with 5% CO2 during imaging. Cells are imaged immediately following the wash step and for up to 2.5 hours after dye addition on a Zeiss spinning disk microscope at 100× with the following general settings: 405 nm at 0.28 mW, 200 ms exposure; 638 nm at 2.4 mW, 200 ms exposure; acquiring each channel at each z-step.
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ES cells and neural differentiation are detailed elsewhere [38 (link)]. The LC1 and other ES cell–derived NS cells were routinely generated by re-plating d 7 adherent neural differentiation cultures (typically 2–3 × 106 cells into a T75 flask) on uncoated plastic in NS-A medium (Euroclone, Milan, Italy) supplemented with modified N2 [36 (link)] and 10 ng/ml of both EGF and FGF-2 (NS expansion medium). Over 3–5 d, cells formed aggregates that, after harvesting and sedimentation to remove debris, subsequently attached to fresh plastic and outgrew NS cells. After addition of 0.5 μg/ml of puromycin to differentiating adherent cultures at d 7, 46C-NS cells were generated. Cells were re-plated 3 d later into an uncoated T75 flask in N2B27 media with 10 ng/ml of both EGF and FGF-2 (Peprotech, Rocky Hill, New Jersey, United States) in the absence of puromycin. To derive clonal lines, including NS-5, single cells were plated into 96-well microwell plates (Nalge Nunc International, Rochester, New York, United States) by limiting dilution. and the presence of one cell per well was scored 1 h after plating.
For derivation directly from foetal CNS, primary cultures were generated using standard protocols from cortex or striatum of E16.5 mouse embryos and subsequently allowed to attach on flasks treated with 0.1% gelatin. Outgrowing cells were then expanded on gelatin using NS expansion medium. Clonal derivatives of the cortical line Cor-1 were established by plating at very low density (1,000 cells per 9-cm plate) and expanding individual colonies.
For derivation from established neurospheres, derived from E14 foetal brain and maintained for 40 passages in EGF plus FGF-2, cultures were dissociated to single cells using Accutase (Sigma, St. Louis, Missouri, United States) and plated at 104 cells/ml on gelatin-coated culture flasks in NS expansion medium.
For passaging established NS cell lines, we routinely used trypsin/EDTA or PBS and split cells 1:3 to 1:5 every 2–3 d. For astrocyte differentiations, NS cells were re-plated onto 4-well plates at 1 × 105 cells/well in NS-A medium supplemented with 1% fetal calf serum or 10 ng/ml BMP4 (R&D Systems, Minneapolis, Minnesota, United States). For neuronal differentiation, 5 × 104 NS cells were plated into poly-ornithine/laminin treated wells in NS-A supplemented with FGF-2 alone. After 7 d, the media was switched to NS-A supplemented with B27 (GIBCO, San Diego, California, United States) without growth factor. Half of the medium was exchanged every 2–3 d during the differentiation. For clonal differentiation, 1,000 cells from NS-5 or Cor-1, cultures were plated in 10-cm plates pre-treated with laminin, expanded for 12 d in EGF/FGF-2, and differentiated in situ as above. For electrophysiological studies, 1.5 × 105 NS cells were plated into poly-L-ornithine-treated 35-mm dishes in NS-A medium supplemented with N2 and B27 (both at 0.5%) and FGF-2 (5 ng/ml). After 7 d, the medium was switched to the mix NS-A:Neurobasal (1:1), supplemented with B27 (GIBCO) without growth factors. To sustain neuronal maturation, after a further 7 d, the medium was switched to the mix NS-A:Neurobasal (1:3) supplemented with B27 (GIBCO) and brain derived neurotrophic factor (20 ng/ml) and nerve growth factor (R&D Systems; 50 ng/ml). Throughout neuronal differentiation, half of the medium was replaced every 2–3 d. Further details of NS cell derivation, propagation, and differentiation are provided inProtocol S1 .
For derivation directly from foetal CNS, primary cultures were generated using standard protocols from cortex or striatum of E16.5 mouse embryos and subsequently allowed to attach on flasks treated with 0.1% gelatin. Outgrowing cells were then expanded on gelatin using NS expansion medium. Clonal derivatives of the cortical line Cor-1 were established by plating at very low density (1,000 cells per 9-cm plate) and expanding individual colonies.
For derivation from established neurospheres, derived from E14 foetal brain and maintained for 40 passages in EGF plus FGF-2, cultures were dissociated to single cells using Accutase (Sigma, St. Louis, Missouri, United States) and plated at 104 cells/ml on gelatin-coated culture flasks in NS expansion medium.
For passaging established NS cell lines, we routinely used trypsin/EDTA or PBS and split cells 1:3 to 1:5 every 2–3 d. For astrocyte differentiations, NS cells were re-plated onto 4-well plates at 1 × 105 cells/well in NS-A medium supplemented with 1% fetal calf serum or 10 ng/ml BMP4 (R&D Systems, Minneapolis, Minnesota, United States). For neuronal differentiation, 5 × 104 NS cells were plated into poly-ornithine/laminin treated wells in NS-A supplemented with FGF-2 alone. After 7 d, the media was switched to NS-A supplemented with B27 (GIBCO, San Diego, California, United States) without growth factor. Half of the medium was exchanged every 2–3 d during the differentiation. For clonal differentiation, 1,000 cells from NS-5 or Cor-1, cultures were plated in 10-cm plates pre-treated with laminin, expanded for 12 d in EGF/FGF-2, and differentiated in situ as above. For electrophysiological studies, 1.5 × 105 NS cells were plated into poly-L-ornithine-treated 35-mm dishes in NS-A medium supplemented with N2 and B27 (both at 0.5%) and FGF-2 (5 ng/ml). After 7 d, the medium was switched to the mix NS-A:Neurobasal (1:1), supplemented with B27 (GIBCO) without growth factors. To sustain neuronal maturation, after a further 7 d, the medium was switched to the mix NS-A:Neurobasal (1:3) supplemented with B27 (GIBCO) and brain derived neurotrophic factor (20 ng/ml) and nerve growth factor (R&D Systems; 50 ng/ml). Throughout neuronal differentiation, half of the medium was replaced every 2–3 d. Further details of NS cell derivation, propagation, and differentiation are provided in
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2-Mercaptoethanol
4-(5-benzo(1,3)dioxol-5-yl-4-pyridin-2-yl-1H-imidazol-2-yl)benzamide
accutase
Amino Acids, Essential
Cardiac Arrest
Cell Culture Techniques
Cells
Chimera
Culture Media, Conditioned
dispase
DNA, Complementary
dorsomorphin
Fibroblast Growth Factor 2
Gelatins
Glutamine
Human Embryonic Stem Cells
Hyperostosis, Diffuse Idiopathic Skeletal
Induced Pluripotent Stem Cells
Insulin
Laminin
matrigel
Mus
Nervousness
Neuroepithelial Cells
Neurogenesis
Neurons
NKX2-1 protein, human
noggin protein
Oligonucleotide Primers
Ornithine
Penicillins
Pluripotent Stem Cells
Poly A
purmorphamine
Reverse Transcriptase Polymerase Chain Reaction
Streptomycin
Transforming Growth Factor beta
trizol
Vitamin A
Most recents protocols related to «Accutase»
After the NPCs had fully differentiated (3 weeks), the cells were detached by accutase 3 min. the cells were collected and centrifuged at regular spin speed. The plates were coated with Matrigel and the cells were added in the culture medium (DMEM/ F12, 10% FBS, and 1% Penicillin). The cells were incubated until confluent. The cells were detached by using accutase and centrifuged at a spin speed of 300 g for 5 min. The step was repeated two times (S1).
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Neurospheres were centrifuged, either resuspended with accutase as single cells or kept as small neurospheres and irradiated using a Cs-137 source (GSR Cs137/C, Gamma Service Medical GmbH) at the indicated doses. For the TMZ treatment, neurospheres were centrifuged, resuspended with accutase as single cells, and seeded at optimal density depending on the experiment. TMZ was diluted in DMSO and used at indicated concentrations.
For combinatorial experiments (γ-irradiation and TMZ) cells were first irradiated as indicated above before seeded in the presence of TMZ at indicated concentrations.
For combinatorial experiments (γ-irradiation and TMZ) cells were first irradiated as indicated above before seeded in the presence of TMZ at indicated concentrations.
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This was done as previously described11 (link). Briefly, the skin from euthanized neonatal mice of indicated genotypes was incubated with 250 units/mL dispase (Roche, cat. #04942078001) in KSFM medium (Thermo Fisher, cat. #17005-042) for 16 h at 4 °C to separate the epidermis from the dermis. The epidermis was then treated with accutase (CELLnTEC, cat. #CnT-accutase-100) for 30 min at 37 °C to release the keratinocytes. The isolated cells were then cultured in KSFM medium supplemented with 20 nM CaCl2, 25 μg/mL BPE and 0.25 ng/mL EGF (Thermo Fisher, cat. #37000-015).
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Tumor tissue from mice were physically cut with scalpels into as small pieces as possible and dissociated with Accutase at 37°C for 10 min. Cells were then plated in 6-well plates in a modified version of NBE media40 (link) consisting of Neurobasal media (Thermo Fisher), 1x Glutamax (Thermo Fisher), 1x N2 supplement (Thermo Fisher), 1x B27 without vitamin A supplement (Thermo Fisher), human recombinant FGF2 and EGF (both from Peprotech at 20 ng/mL). Doxycycline (Sigma Aldrich) was added at 1ug/mL to maintain expression of inducible MYCN. Media is changed every 2–3 days and tumor cells were passaged using Accutase.
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Top products related to «Accutase»
Sourced in United States, Germany, United Kingdom, France, Italy, Switzerland, Sao Tome and Principe, Macao, Denmark
Accutase is a laboratory reagent that is used for the enzymatic dissociation and harvesting of adherent cells in cell culture. It is a proprietary mixture of proteolytic and collagenolytic enzymes that effectively detaches cells from the culture surface without affecting cell viability or cell surface receptors.
Sourced in United States, United Kingdom, Germany, Switzerland, Canada, Belgium, China, Sweden, Japan
Accutase is a cell detachment solution designed for the gentle dissociation of adherent cells. It contains a mixture of proteolytic and collagenolytic enzymes that effectively disrupt cell-cell and cell-matrix adhesions, allowing for the easy harvesting and passaging of a variety of cell types.
Sourced in Canada, United States, France, Germany, United Kingdom, China
Accutase is a cell detachment solution used for the gentle dissociation of adherent cells from cell culture surfaces. It is a mixture of proteolytic and collagenolytic enzymes designed to effectively detach cells while minimizing damage to cell surface proteins and preserving cell viability.
Sourced in United States
Accutase is a cell detachment solution designed for the gentle dissociation of adherent cells. It is a mixture of enzymes that effectively breaks down the extracellular matrix and cell-cell junctions, allowing for the efficient harvesting of cells while maintaining their viability and functionality.
Sourced in United States, Germany, United Kingdom, Italy, Canada, Sweden
StemPro Accutase is a cell dissociation reagent used for the gentle dissociation of adherent cells, including stem cells and other sensitive cell types. It is a non-enzymatic, animal-component-free solution designed to maintain cell viability and preserve cell surface epitopes.
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DMEM/F12 is a cell culture medium developed by Thermo Fisher Scientific. It is a balanced salt solution that provides nutrients and growth factors essential for the cultivation of a variety of cell types, including adherent and suspension cells. The medium is formulated to support the proliferation and maintenance of cells in vitro.
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Penicillin/streptomycin is a commonly used antibiotic solution for cell culture applications. It contains a combination of penicillin and streptomycin, which are broad-spectrum antibiotics that inhibit the growth of both Gram-positive and Gram-negative bacteria.
Sourced in United States, Germany, United Kingdom, France, Switzerland, Canada, Japan, Australia, China, Belgium, Italy, Denmark, Spain, Austria, Netherlands, Sweden, Ireland, New Zealand, Israel, Gabon, India, Poland, Argentina, Macao, Finland, Hungary, Brazil, Slovenia, Sao Tome and Principe, Singapore, Holy See (Vatican City State)
GlutaMAX is a chemically defined, L-glutamine substitute for cell culture media. It is a stable source of L-glutamine that does not degrade over time like L-glutamine. GlutaMAX helps maintain consistent cell growth and performance in cell culture applications.
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Fetal Bovine Serum (FBS) is a cell culture supplement derived from the blood of bovine fetuses. FBS provides a source of proteins, growth factors, and other components that support the growth and maintenance of various cell types in in vitro cell culture applications.
Sourced in United States, China, Germany, United Kingdom, Canada, Japan, France, Netherlands, Montenegro, Switzerland, Austria, Australia, Colombia, Spain, Morocco, India, Azerbaijan
Matrigel is a complex mixture of extracellular matrix proteins derived from Engelbreth-Holm-Swarm (EHS) mouse sarcoma cells. It is widely used as a basement membrane matrix to support the growth, differentiation, and morphogenesis of various cell types in cell culture applications.
More about "Accutase"
Accutase is a versatile and gentle enzymatic cell dissociation reagent that is widely used in stem cell research, cell culture, and regenerative medicine applications.
It is a mixture of proteolytic and collagenolytic enzymes that can effectively detach adherent cells from culture surfaces while maintaining their viability and surface antigen expression.
Unlike traditional dissociation methods like trypsin, Accutase is known for its milder and more gentle dissociation properties, making it a suitable alternative for sensitive cell types.
This makes it particularly useful in applications where preserving cell integrity and function is crucial, such as in stem cell research and culture.
StemPro Accutase is a specific formulation of Accutase that is optimized for stem cell work, providing a reliable and consistent solution for researchers.
Alongside Accutase, other commonly used cell culture components include DMEM/F12 media, Penicillin/Streptomycin antibiotics, GlutaMAX supplements, and Fetal Bovine Serum (FBS) for cell growth and maintenance.
Matrigel is also often used as a substrate for stem cell culture and differentiation.
By leveraging PubCompare.ai's AI-driven platform, researchers can easily locate and compare Accutase protocols from a wide range of literature sources, including journal articles, preprints, and patents.
This streamlines the research process and helps users identify the most suitable protocols and products for their specific needs, ultimately optimizing their results and saving valuable time.
It is a mixture of proteolytic and collagenolytic enzymes that can effectively detach adherent cells from culture surfaces while maintaining their viability and surface antigen expression.
Unlike traditional dissociation methods like trypsin, Accutase is known for its milder and more gentle dissociation properties, making it a suitable alternative for sensitive cell types.
This makes it particularly useful in applications where preserving cell integrity and function is crucial, such as in stem cell research and culture.
StemPro Accutase is a specific formulation of Accutase that is optimized for stem cell work, providing a reliable and consistent solution for researchers.
Alongside Accutase, other commonly used cell culture components include DMEM/F12 media, Penicillin/Streptomycin antibiotics, GlutaMAX supplements, and Fetal Bovine Serum (FBS) for cell growth and maintenance.
Matrigel is also often used as a substrate for stem cell culture and differentiation.
By leveraging PubCompare.ai's AI-driven platform, researchers can easily locate and compare Accutase protocols from a wide range of literature sources, including journal articles, preprints, and patents.
This streamlines the research process and helps users identify the most suitable protocols and products for their specific needs, ultimately optimizing their results and saving valuable time.