Colonies were transferred to ultra-low attachment plates (Corning) to generate embryoid bodies (EB)17 (link). EBs were treated with neural induction medium [NIM: DMEM/F12/glutamax/N2/B27 (Invitrogen) and pen/strep)] for 1 week. Cells were transferred to polyornithine/laminin (Invitrogen)-coated plates. After 7 d, neural rosettes were dissected from the EBs and expanded in NIM containing 20 ng/ml FGF2 (Preprotech). Rosettes were dispersed to form NPCs and passaged every 5–7 d. To obtain mature neurons, NPCs were treated with NIM with 20 ng/ml BDNF (Preprotech), 20 ng/ml GDNF (Peprotech), 1 mm dibutyryl-cyclic AMP (Sigma–Aldrich) and 200 nm ascorbic acid (Sigma–Aldrich), and cultured with weekly half media changes. Neurons used in experiments were differentiated for 6–8 wk. At least two clones were independently grown for each line. In studies that required multiple wells of the same line (e.g. PCR, temperature cycles) it was not always possible to grow sufficient numbers of cells for experiments and in cases, some lines were not included. A detailed accounting of cells allocated for each experiment is provided (Table S2 –S3 ).
Polyornithine laminin
Manufactured by Thermo Fisher Scientific
Sourced in United States
Polyornithine/laminin is a substrate coating that enhances cell attachment and growth. It is a combination of the positively charged polypeptide polyornithine and the extracellular matrix protein laminin.
Automatically generated - may contain errors
Lab products found in correlation
Ultra low attachment plate, by Corning (2 mentions)
Fibroblast growth factor 2 (fgf2), by Thermo Fisher Scientific (2 mentions)
Brain derived neurotrophic factor (bdnf), by Thermo Fisher Scientific (2 mentions)
Ascorbic acid, by Merck Group (2 mentions)
Dmem f12 glutamax n2 b27, by Thermo Fisher Scientific (2 mentions)
Dibutyryl cyclic amp, by Merck Group (2 mentions)
Nunclon plate, by Thermo Fisher Scientific (2 mentions)
Glutamax, by Thermo Fisher Scientific (2 mentions)
Neurobasal medium, by Thermo Fisher Scientific (2 mentions)
Ultra low attachment 96 well plate, by Corning (1 mentions)
5 protocols using polyornithine laminin
1
Directed Differentiation of Pluripotent Stem Cells to Neurons
2
Cerebral Organoid and Neurosphere Generation from iPSCs
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Cerebral organoids were generated from iPSC as previously described (18 (link)). For control lines, EBs were generated from 9000 cells each. For patient lines, EBs were generated from 9000 and 18 000 cells with no visible difference in size or development. Two control- and two SPG11-iPSC lines (SPG11-1 and SPG11-2) were analyzed. Controls were grouped. Cerebral organoids were cultured for 40 to 61 days (6 and 9 weeks) at 37°C under 5% CO2 and atmospheric oxygen. For the rescue experiment, control- and SPG11 organoids were treated with 1 μM of the GSK3 blockers CHIR99021 and tideglusib and kept in culture for 40 to 60 days (6 and 9 weeks). Compounds were replaced every 2–3 days (Supplementary Material, Fig. S4D ).
For the neurosphere assay (3 (link)), NPCs were kept in suspension in 96 well ultra-low attachment plates (Corning, Amsterdam, the Netherlands) for 72 h at a density of 5 × 104 cells/cm2 under proliferative conditions. For the analysis of NPC migration, neurospheres grew attached to polyornithine/laminin (Invitrogen, Carlsbad, California, United States) coated coverslips for 48 h (Fig. 2A ). Spheres generated from six SPG11-NPC lines (SPG11-1, SPG11-2 and SPG11-3) were compared to controls.
For the neurosphere assay (3 (link)), NPCs were kept in suspension in 96 well ultra-low attachment plates (Corning, Amsterdam, the Netherlands) for 72 h at a density of 5 × 104 cells/cm2 under proliferative conditions. For the analysis of NPC migration, neurospheres grew attached to polyornithine/laminin (Invitrogen, Carlsbad, California, United States) coated coverslips for 48 h (
3
Isolation and Culture of Murine Spinal Motor Neurons
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Murine embryonic spinal MNs were isolated and cultured as previously described (Wiese et al., 2010 (link)). Briefly, after dissection of the ventrolateral part of E12.5 embryos, spinal cord tissues were incubated for 15 min in 0.05% trypsin in HBSS. Cells were triturated and incubated in Neurobasal medium (Invitrogen), supplemented with 1× Glutamax (Invitrogen) on Nunclon plates (Nunc) precoated with antibodies against the p75 NGF receptor (MLR2; kind gift of Robert Rush, Flinders University, Adelaide, Australia) for 45 min. Plates were washed with Neurobasal medium, and the remaining MNs were recovered from the plate with depolarization solution (0.8% NaCl, 35 mM KCl and 2 mM CaCl2) and collected in full medium (2% horse serum and 1× B27 in Neurobasal medium with 1× Glutamax). After counting, the cell number was adjusted to 1,000 in 100 µl, and 1,000 cells were plated per well on four-well dishes (Greiner; Cellstar) precoated with poly-ornithine/laminin (Invitrogen). Cells were cultured in the presence of brain-derived neurotrophic factor. Axon length was quantified after 7 d in vitro.
4
Isolation and Culture of Murine Embryonic Spinal Motoneurons
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Murine embryonic spinal motoneurons were isolated and cultured as described56 (link). Briefly, after dissection of the ventrolateral part of E12.5 embryos, spinal cord tissues were incubated for 15 min in 0.05% trypsin in Hank’s balanced salt solution. Cells were triturated and incubated in Neurobasal medium (Invitrogen), supplemented with 1× Glutamax (Invitrogen) on Nunclon plates (Nunc) pre-coated with antibodies against the p75 NGF receptor (MLR2, kind gift of Robert Rush, Flinders University, Adelaide, Australia) for 45 min. Plates were washed with Neurobasal medium, and the remaining motoneurons were recovered from the plate with depolarization solution (0.8% NaCl, 35 mM KCl and 2 mM CaCl2) and collected in full medium (2% horse serum, 1× B27 in Neurobasal medium with 1× Glutamax). After counting, cell number was adjusted to 1000 in 100 µl, and 1000 cells were plated on four-well dishes (Greiner, Cellstar) pre-coated with poly-ornithine/laminin (Invitrogen). Cells were cultured in the presence of the neurotrophic factor BDNF. For survival assays, cells were counted 4 h after plating to find the total number of plated cells. Cells were counted again after 5 and after 7 days in vitro (DIV).
For lentiviral transduction, motoneurons were incubated with viral particles for 10 min at RT directly before plating.
For lentiviral transduction, motoneurons were incubated with viral particles for 10 min at RT directly before plating.
5
Directed Differentiation of Pluripotent Stem Cells to Neurons
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