CytoTune™-iPS 2.0 Sendai Reprogramming 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. -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. -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

Cadaver donor globes from patients with AMD as well as age-matched unaffected individuals were received from the National Disease Research Interchange (Philadelphia, PA, USA) and the Eye-Bank for Sight Restoration, Inc. (New York, NY, USA), and the ocular tissues used to establish either cornea-associated fibroblasts or RPE cells for production of hiPSCs. The RPE cells were isolated and grown to establish a functional RPE monolayer (Blenkinsop et al., 2015; Blenkinsop et al., 2013). To derive hiPSC from these cells, either Sendai virus-based (CytoTune -iPS Sendai Reprogramming kit; Cat. # A13780-01; Thermo Fisher) or mRNA-based (mRNA Reprogramming kit; Cat. # 00-0071; Stemgent) was used (i.e. non-integrating reprogramming methods) according to manufacturer’s instructions. Approximately three weeks after the transduction with Sendai virus or mRNA transfection, colonies with the typical iPSC morphology appeared. Cells were live-stained with directly conjugated AlexaFluor 488-anti TRA-1-60 (Cat. # 560173; BD), positive colonies were then hand-picked and sub-cloned in order to be expanded and characterized. In general, three clones were generated and characterized per hiPSC line.

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Manufacturer protocol

Download the product protocol from Thermo Fisher Scientific for CytoTune™-iPS 2.0 Sendai Reprogramming Kit below.

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