For experiments in Fig. 3 performed at University of Pennsylvania, primary neuronal cultures were prepared from E16–18 CD1 mouse embryos. Dissociated neurons were plated onto poly-d -lysine-coated optical-bottom 96-well plates (ViewPlate; Perkin Elmer) and 60,000 cells cm−2. Cultures were maintained in the Neurobasal medium supplemented with B27 (Invitrogen) that was replenished every 5 days. Sonicated PFFs diluted in sterile PBS without Ca2+/Mg2+ (Corning) were added at 7 days in vitro (DIV) at the indicated concentrations. Cells were fixed in 4% paraformaldehyde at 19 DIV and co-labeled using antibodies against phosphorylated at serine 129 (mouse monoclonal, clone 81A, 1:10,000; CNDR), NeuN (mouse monoclonal, clone A60; Millipore) and microtubule-associated protein 2 (rabbit, 17028, 1:2,000; CNDR). Alexa Fluor-conjugated secondary antibodies mouse IgG1, IgG2a or rabbit IgG were used to visualize staining. Images were obtained using an InCell2200 scanner (GE Life Sciences).
Ca2 mg2
Manufactured by Corning
The Ca2+/Mg2+ lab equipment is a device used for the detection and measurement of calcium and magnesium ions in various samples. It provides accurate and reliable data on the levels of these important ions, which are essential for numerous biological processes.
Automatically generated - may contain errors
Lab products found in correlation
Neurobasal a medium, by Thermo Fisher Scientific (2 mentions)
Recombinant human lif, by Merck Group (2 mentions)
Ack rbc lysis buffer, by Thermo Fisher Scientific (2 mentions)
Dnase, by Worthington (2 mentions)
Bovine serum albumin (bsa), by Gemini Bio (2 mentions)
Matrigel, by Corning (2 mentions)
Intesticult organoid growth medium, by STEMCELL (2 mentions)
Antibiotic antimycotic, by Thermo Fisher Scientific (2 mentions)
Viewplate, by PerkinElmer (1 mentions)
Penicillin streptomycin cocktail, by Merck Group (1 mentions)
Z2 particle counter, by Beckman Coulter (1 mentions)
Trypsin edta solution, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Poly d lysine, by Merck Group (1 mentions)
Glutamax, by Thermo Fisher Scientific (1 mentions)
Neurobasal medium, by Thermo Fisher Scientific (1 mentions)
Bioruptor plus, by Diagenode (1 mentions)
Gentle cell dissociation reagent (gcdr), by STEMCELL (1 mentions)
Nylon cell strainer, by BD (1 mentions)
70 mm nylon cell strainer, by BD (1 mentions)
7 protocols using ca2 mg2
1
Primary Neuronal Culture and α-Synuclein Seeding
2
Cell Culture with Strict Parameters
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Cells were cultured in 25 cm2 polystyrene culture flasks (Eppendorf) in DMEM medium with high glucose (4,500 mg/l) (Sigma-Aldrich), supplemented with 10% fetal bovine serum (Gibco) and 1% antibiotics: penicillin/streptomycin cocktail (Sigma-Aldrich). The cultures were grown under strictly controlled conditions: 5% CO2, temperature 37 °C. The medium was changed every two days and the cells passaged when the confluence reached 80%. For that purpose the cells were washed with PBS without Ca2+/Mg2+ (Corning), harvested with 0.25% Trypsin-EDTA solution (Gibco), resuspended in fresh medium, counted using Beckman Z2 particle counter and seeded into plastic culture dishes.
3
Preparation and Treatment of Primary Neuronal Cultures
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Primary neuronal cultures were prepared from CD1 embryos on E16–18, as previously described58 (link). Primary mouse neuron protocol was approved by the University of Pennsylvania Institutional Animal Care and Use Committee. Tissue culture plates and coverslips were coated with Poly-d -lysine (Sigma) before addition of cells. Neurons were plated in 96-well plates (60,000 cells/cm2). Cultures were maintained in Neurobasal medium supplemented with B27 and Glutamax (all from Invitrogen).
PFF treatment was performed at 7 DIV and cultures were incubated for a further 12 days prior to fixation and analysis. Briefly, stock αSN PFFs were diluted in sterile DPBS (without Ca2+/Mg2+; Corning)—8 μL of aSyn + 392 μL of DPBS into 1.5 mL Eppendorf tubes—and sonicated with a bath sonicator (Bioruptor Plus, Diagenode) for 10 cycles at high power (30 s on, 30 s off, at 10 °C). Sonicated PFFs were then further diluted to the indicated final concentration in neuronal media and added to cultures. PFF concentrations are expressed as the total equivalent αSN monomer content in the preparation. Cultures were treated with 200 nM PFFs unless otherwise noted. Compounds were initially diluted in neuronal media before being added to the PFF neuronal media solution at a final concentration of 1–10 μM.
PFF treatment was performed at 7 DIV and cultures were incubated for a further 12 days prior to fixation and analysis. Briefly, stock αSN PFFs were diluted in sterile DPBS (without Ca2+/Mg2+; Corning)—8 μL of aSyn + 392 μL of DPBS into 1.5 mL Eppendorf tubes—and sonicated with a bath sonicator (Bioruptor Plus, Diagenode) for 10 cycles at high power (30 s on, 30 s off, at 10 °C). Sonicated PFFs were then further diluted to the indicated final concentration in neuronal media and added to cultures. PFF concentrations are expressed as the total equivalent αSN monomer content in the preparation. Cultures were treated with 200 nM PFFs unless otherwise noted. Compounds were initially diluted in neuronal media before being added to the PFF neuronal media solution at a final concentration of 1–10 μM.
4
Medulloblastoma Tumor Tissue Dissociation
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A medulloblastoma tissue sample was obtained after appropriate patient consent at the Lucille Packard Children’s Hospital (Stanford, CA), in accordance with Institutional Review Board protocols (Protocol ID #: 12625; IRB #: 4593 (Panel: 5)). Pathology of the tumor was confirmed upon histopathologic analysis by institutional neuropathologists prior to further analysis.
The tumor sample was mechanically dissociated in a solution containing Hank’s Balanced Salt Solution (HBSS), nonessential amino acids, sodium pyruvate, sodium bicarbonate, HEPES, Glutamax-1, antibiotic-antimycotic, DNase, and collagenase IV. All media components were from Cellgro, except for DNase and collagenase IV, which were from Worthington. The suspension was washed two times with HBSS, filtered through a 70-μm strainer and resuspended in a 0.9 M sucrose solution in HBSS without Ca2+/Mg2+ (Cellgro) to remove debris and dead cells. The cells were treated with ACK/RBC lysis buffer (Gibco), then washed twice in phosphate buffered saline (PBS). Cells were plated in tumor stem media (TSM) consisting of Neurobasal-A medium (Invitrogen), B27-A (Invitrogen), human bFGF (20 ng/ml) (Shenandoah Biotech), human EGF (20 ng/ml) (Shenandoah Biotech), human recombinant LIF (20 ng/ml) (Millipore), and heparin (10 ng/ml).
The tumor sample was mechanically dissociated in a solution containing Hank’s Balanced Salt Solution (HBSS), nonessential amino acids, sodium pyruvate, sodium bicarbonate, HEPES, Glutamax-1, antibiotic-antimycotic, DNase, and collagenase IV. All media components were from Cellgro, except for DNase and collagenase IV, which were from Worthington. The suspension was washed two times with HBSS, filtered through a 70-μm strainer and resuspended in a 0.9 M sucrose solution in HBSS without Ca2+/Mg2+ (Cellgro) to remove debris and dead cells. The cells were treated with ACK/RBC lysis buffer (Gibco), then washed twice in phosphate buffered saline (PBS). Cells were plated in tumor stem media (TSM) consisting of Neurobasal-A medium (Invitrogen), B27-A (Invitrogen), human bFGF (20 ng/ml) (Shenandoah Biotech), human EGF (20 ng/ml) (Shenandoah Biotech), human recombinant LIF (20 ng/ml) (Millipore), and heparin (10 ng/ml).
5
Medulloblastoma Tumor Tissue Dissociation
6
Isolation and Culture of Intestinal Organoids
7
Intestinal Organoid Isolation and Culture
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Intestines were washed in ice-cold PBS (Mg2+/Ca2+ (Corning, cat # 21-031-CM), containing 2% BSA (Gemini Bio-products, cat #900–208) and 2% antibiotic-antimycotic (Gibco, cat #15240–062). Crypts and villi were exposed by dicing the intestines into small pieces (1–2 cm long), followed by extensive washes to remove contaminants. Then, a gentle cell dissociation reagent (Stem cell technologies, cat #7174) was used according to the manufacturer’s instructions. Briefly, intestinal pieces were incubated on a gently rotating platform for 15 min. After that, the gentle cell dissociation reagent was removed and the intestines were washed 3 times with a PBS wash buffer with vigorous pipetting. The first and second fractions that usually contain loose pieces of mesenchyme and villi were not used. Fractions three and four containing the intestinal crypts were collected and pooled. Isolated crypts were filtered through a 70mm nylon cell strainer (Falcon, cat #352350). Crypts were counted, then embedded in Matrigel (Corning, growth factor reduced, cat #354230), and cultured in Intesticult organoid growth medium (Stem cell technologies, cat #6005). For mouse colon organoids, additional Wnt3a (300 ng/μL, R&D, cat #5036-WN-010) was added. Intestinal organoids used in this study were generated from WT and APCmin/+ mice at 37°C, 5% CO2.
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