Generating Cortical Interneuron Precursors From Mouse Embryonic Stem Cells
DEŞİFRE METNİ
Start growing the cells as embryoid bodies by adding 75,000 cells per milliliter in KSRN2 media in the non-adherent tissue culture dishes and incubate them at 37 degrees Celsius. On DD1, prepare for cell landing by coating the tissue culture-treated dishes with poly-L-lysine for at least one hour or overnight at 37 degrees Celsius.
On DD2, aspirate the poly-L-lysine and coat the plates with laminin overnight at 37 degrees Celsius. On DD3, before beginning EB dissociation, aspirate laminin, and allow the plates to completely dry in a tissue culture hood.
Afterward, transfer the EBs with the media into a 15-milliliter tube and centrifuge for three to four minutes at 15 times g or until the EBs have pelleted. Aspirate the media and add 3 milliliters of cell detachment solution containing DNase. Then, incubate the sample at 37 degrees Celsius for 15 minutes.
Gently flick the tube every three minutes to aid in EB dissociation. Once the EBs are no longer visible or 15 minutes have elapsed, add 6 milliliters of KSRN2 containing DNase and centrifuge for five minutes at 200 times g. Then, plate the cells in KSRN2 containing LDN193189, XAV939, and the ROCK inhibitor Y27632 at 4.5 to 5 x 104 cells per centimeter squared.
