HB9, a homeobox gene, is expressed in embryonic MNs early after differentiation from neuronal progenitors.18 , 19 An Hb9‐eGFP ES cell line on a pure C57Bl6 background, denoted as HBGB6, was generously provided by Dr. Craig Cox (Jackson, ME) and was used to generate MNs. ESCMNs were generated by treating free‐floating clusters of ES cells known as embryonic bodies (EB) with retinoic acid (1 μmol/L; Sigma Aldrich, Oakville, ON, Canada) and smoothened agonist (500 nmol/L; Enzo Life Sciences, Farmingdale, NY, USA) as described previously.7 , 13 , 14 Differentiated EBs were enzymatically dissociated in Tryple express (ThermoFisher Scientific, Ottawa, ON, Canada) with 0.01% (w/v) DNaseI (Sigma‐Aldrich). The obtained single cell suspension was resuspended in DFK10.14 For in vitro experiments, 105 dissociated cells were plated onto growth factor reduced matrigel (BD Biosciences, Rockville, MD, USA)‐coated glass coverslips (ThermoFisher Scientific). In vitro cells were maintained in DFK10 supplemented with 10 ng/mL GDNF (Millipore, Etobicoke, ON, Canada) and 10 ng/mL CNTF (ThermoFisher Scientific). Media was changed every other day.
Smoothened agonist
Manufactured by Enzo Life Sciences
Sourced in Canada
Smoothened agonist is a chemical compound that acts as an agonist for the Smoothened receptor, a key component of the Hedgehog signaling pathway. It plays a role in the regulation of various cellular processes.
Automatically generated - may contain errors
Lab products found in correlation
Retinoic acid, by Merck Group (2 mentions)
Neurobasal medium, by Thermo Fisher Scientific (2 mentions)
Dmem f12, by Thermo Fisher Scientific (2 mentions)
Coated glass coverslips, by Thermo Fisher Scientific (1 mentions)
Tryple express, by Thermo Fisher Scientific (1 mentions)
Dnase 1, by Merck Group (1 mentions)
Growth factor reduced matrigel, by BD (1 mentions)
Neurotrophin 3 nt 3, by R&D Systems (1 mentions)
Brain derived neurotrophic factor bdnf, by R&D Systems (1 mentions)
Y 27632, by Nacalai Tesque (1 mentions)
2 protocols using smoothened agonist
1
Generation of Hb9-eGFP Motor Neurons
2
Efficient Motor Neuron Differentiation from ESCs/iPSCs
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
ESCs/iPSCs were treated with CTK dissociation solution for 2 min and feeder cells were removed with PBS. Then ESCs/iPSCs were dissociated to single cells with Accumax, and they were transferred onto Matrigel-coated plates with MN medium containing a 1:1 mixture of Neurobasal medium (Thermo Fisher Scientific) and DMEM/F12 (Thermo Fisher Scientific), supplemented with 0.5% N2 (Thermo Fisher Scientific), 1% B27 (Thermo Fisher Scientific), 1 μM retinoic acid (Sigma-Aldrich), 1 μM smoothened agonist (Enzo Life Sciences), 10 ng/mL brain-derived neurotrophic factor (BDNF; R&D Systems), 10 ng/mL glial cell-derived neurotrophic factor (GDNF; R&D Systems), 10 ng/mL neurotrophin-3 (NT-3; R&D Systems), and 10 μM Y-27632 (Nacalai Tesque). At the same time, the ESCs/iPSCs were infected with SeV-L-N-I; SeV-L-N-I-EGFP; or combinations of SeV-L, SeV-N, and SeV-I (ID Pharma) on day 0. MOIs were 5 or 10. The transduction of SeV vectors to human ESCs/iPSCs was conducted just once. The number of cells per well was 5.0 × 104 in 96-well plates and 1.0 × 106 in 12-well plates. The medium was changed to MN medium without Y-27632 on day 1 and then changed every 3 days.
For phenotype assays, cells were treated with Accumax plus 10 μM Y-27632 and transferred onto poly-L-lysine- and Matrigel-coated glass dishes on day 7. For immunocytochemistry and qPCR analyses, cells were assessed on day 14.
For phenotype assays, cells were treated with Accumax plus 10 μM Y-27632 and transferred onto poly-L-lysine- and Matrigel-coated glass dishes on day 7. For immunocytochemistry and qPCR analyses, cells were assessed on day 14.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!