STEMdiff™ SMADi Neural Induction Kit

Protocol tips

Upstream tips	Protocol tips	Downstream tips
-If STEMdiff™ Neural Induction Medium is received thawed, immediately place at -20°C or aliquot and store at -20°C. Product performance will not be affected.	-If not used immediately, the cultureware must be sealed to prevent evaporation of the laminin solution (e.g. with Parafilm®) and can be stored at 2 - 8°C for up to 2 weeks after coating. Allow stored coated cultureware to come to room temperature (15 - 25°C) for 30 minutes before plating cells. -If the cultureware surface is not fully coated by the Matrigel® solution, it should not be used for human ES or iPS cell culture. - The incubation time may vary when using different cell lines or other non-enzymatic cell dissociation reagents, therefore dissociation should be monitored under the microscope until the optimal time is determined.

Upstream tips
-If STEMdiff™ Neural Induction Medium is received thawed, immediately place at -20°C or aliquot and store at -20°C. Product performance will not be affected.
Protocol tips
-If not used immediately, the cultureware must be sealed to prevent evaporation of the laminin solution (e.g. with Parafilm®) and can be stored at 2 - 8°C for up to 2 weeks after coating. Allow stored coated cultureware to come to room temperature (15 - 25°C) for 30 minutes before plating cells. -If the cultureware surface is not fully coated by the Matrigel® solution, it should not be used for human ES or iPS cell culture. - The incubation time may vary when using different cell lines or other non-enzymatic cell dissociation reagents, therefore dissociation should be monitored under the microscope until the optimal time is determined.

Publication protocol

A small dermal tissue sample was collected from an excess specimen at the time of a surgical
procedure of the 10-month-old female CFNS proband. Punch biopsies were obtained from the proband’s father and mother. Primary
human fibroblast cultures were established and cultured on plastic culture dishes in DMEM high glucose (Life Technologies)
containing 10% FBS (HyClone), 2 mM L-glutamine, 1 mM sodium pyruvate, 0.1 mM MEM non-essential amino acids, and
penicillin-streptomycin-fungizone. Human iPSCs were generated using episomal reprogramming (Bershteyn et al., 2014; Okita et al.,
2011). Briefly, one microgram of each of the Y4 combination of episomal reprogramming factors (Addgene 27078, 27080, 27082)
was electroporated into 3 x105 fibroblasts (passage 5-6) with the Neon Electroporation Device (Invitrogen) using the 100-uL kit and
conditions of 1650 V, 10 ms, and three pulses. Cells were detached 6 days after electroporation and seeded at 1.5 x 105 cells per 10-cm
dish onto irradiated mouse embryonic fibroblasts (Globalstem). On day 7, media was changed from fibroblast media to KnockOutTM
ESC/hiPSC culture media containing 4 ng/mL bFGF (Life Technologies), and cells were cultured for a further 18-25 days. Colonies
with hiPSC-like morphology were manually selected under a dissecting microscope and subcultured on irradiated MEFs. By passage
four, hiPSCs were transferred to feeder-free conditions and cultured in mTeSR1 medium (STEMCELL Technologies) on dishes
coated with hESC-qualified Matrigel (Corning).

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Manufacturer protocol

Download the product protocol from STEMCELL technologies for STEMdiff™ SMADi Neural Induction Kit below.

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