![]() Cells were then washed one time in PBS and lysed in 50 mM Tris pH 8.0 with 1% SDS and Proteinase K overnight at 37☌. C10424) for 2 hours shaking at room temperature. B10184), and 1X Click-iT additive (ThermoFisher, Cat. Then the following were added in order: 3 mM copper sulfate (Sigma), 50 M biotin azide (ThermoFisher, Cat. For biotin labeling via Click-iT reaction, cells were first washed 1x in PBS, permeabilized with 0.2% Triton-X100/PBS for 10 minutes on ice, and then washed 1x in PBS. Samples were kept on ice for 20 minutes and then stored at -20C until processing.Ĭopper catalyzes azide-alkyne click chemistry. Cold methanol was then added dropwise during slow vortexing to 80% final concentration. We did not detect any differences for experiments where day 3 neurons were thawed or plated immediately following differentiation.Ĭells were washed with PBS, incubated with accutase for 5-10 mins, collected with a cell scrapper, pelleted at 200 × g for 5 minutes and resuspended in cold 0.1% BSA in PBS. 25-950-CQC), and then thawed rapidly at 37☌, followed by removal of FBS/DMSO and plating in i3Neuron Culture Media. ES-009-B) and 10% DMSO (Mediatech Inc., Cat. In some experiments pre-differentiated i3Neurons were frozen on day 3 in 90% fetal bovine serum (Sigma Aldrich, Cat. For i3Neurons cultured beyond 7 days, half media changes were conducted three times per week. Unless otherwise noted, i3Neurons were fed on day 6 during a half media change and harvested on day 7. For ibidi slides used in imaging experiments, 0.2 million neurons per well were plated. For 15 cm plates 30-45 million neurons were plated. 631311).For 10 cm plates used in SAR-seq or CHIP-seq, 12-15 million neurons were plated. L2020-1MG), and 2 µg/mL doxycycline (Clontech, Cat. 450-03), 1 mg/mL mouse laminin (Sigma, Cat. 05790) supplemented with 1x B27 Plus Supplement (ThermoFisher Scientific, Cat. P3655-10MG) coated dishes as follows: Cells were washed with PBS, dissociated with accutase for 10 minutes at 37☌, washed and plated in i3Neuron Culture Media: BrainPhys media (STEMCELL Technologies, Cat. Day 3 cells were replated onto freshly-prepared poly-L-ornithine- (PLO 0.1 mg/ml Sigma, Cat. N2 media was changed once a day for two more days. S1049), and 2 µg/mL doxycycline (Clontech, Cat. 35050061), 1x MEM nonessential amino acids (NEAA) (Thermofisher Scientific, Cat. 17502048), 1x GlutaMAX (Thermofisher Scientific, Cat. 12660012) with N2 supplement (Life Technologies Corporation, Cat. Neurons and iMuscle cells were incubated with 20 M EdU for 18 hours, unless otherwise noted.įor neuronal differentiation, 20-25 million iPSCs were plated on day 0 onto a 15 cm plate in N2 media composed of knockout DMEM/F12 media (Life Technologies Corporation, Cat. GEO help: Mouse over screen elements for information.Ĭell type: neuron differentiated from iPSC
0 Comments
Leave a Reply. |
AuthorWrite something about yourself. No need to be fancy, just an overview. ArchivesCategories |