A skin biopsy was obtained from a MELAS patient. Human fibroblasts were cultured in fibroblast medium (DMEM) supplemented with 10% fetal bovine serum (FBS) (HyClone), 1 mmol/L glutamine (Gibco), 1% non-essential amino acids (NEAA) (Gibco), and 100 IU/mL penicillin/streptomycin (Gibco). To generate iPSCs, fibroblast cells were transfected with a Sendai virus reprogramming kit (Life Technologies, A16517). The transfected cells were then plated onto Matrigel-coated culture dishes according to the manufacturer’s instructions. To remove the Sendai viruses, iPSCs were incubated at 38.5°C for 4 days as instructed. All iPSCs were cultured on Matrigel-coated tissue culture dishes (ES-qualified, BD Biosciences) with mTeSR1 (STEMCELL Technologies) at 37°C and 5% CO2 in a 100% humidified atmosphere incubator. The culture medium was changed daily until the cells were ready for passage or harvest. The cells were passaged every 3–4 days using Accutase (Stemcell Technologies).
Sendai virus reprogramming kit
Manufactured by Thermo Fisher Scientific
The Sendai virus reprogramming kit is a laboratory tool used to generate induced pluripotent stem cells (iPSCs) from various cell types. It provides the necessary components to efficiently introduce the Sendai virus-based reprogramming factors into target cells, enabling the conversion of differentiated cells into a pluripotent state.
Automatically generated - may contain errors
Lab products found in correlation
Mtesr1, by STEMCELL (3 mentions)
Accutase, by STEMCELL (3 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
Non essential amino acids (neaa), by Thermo Fisher Scientific (1 mentions)
Glutamine, by Thermo Fisher Scientific (1 mentions)
Qx200 droplet digital pcr system, by Bio-Rad (1 mentions)
Ddpcr supermix for probes no dutp kit, by Bio-Rad (1 mentions)
Es qualified, by BD (1 mentions)
3 protocols using sendai virus reprogramming kit
1
MELAS Fibroblast-Derived iPSC Generation
2
Generating Mitochondrial Disease iPSCs
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To generate mitochondria diseases iPSCs, endometrium tissues fibroblast cells were transfected with a Sendai virus reprogramming kit (Life Technologies, A16517). The transfected cells were then plated onto Matrigel-coated culture dishes according to the manufacturer's instructions. The iPSCs were cultured on Matrigel-coated tissue culture dishes (ES-qualified, BD Biosciences) with mTeSR1 (STEMCELL Technologies) at 37°C and 5% CO2 in a humidified atmosphere incubator. The iPSCs culture medium was changed daily. The cells were passaged every 3–4 days using Accutase (Stemcell Technologies). The iPSCs conversion to EPS was preformed as previously reported (23 (link)).
3
Mitochondrial Disease iPSC Generation and Droplet Digital PCR
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To generate mitochondria diseases iPSCs, endometrium tissues fibroblast cells were transfected with a Sendai virus reprogramming kit (Life Technologies, A16517). The transfected cells were then plated onto Matrigel-coated culture dishes according to the manufacturer's instructions.
The iPSCs were cultured on Matrigel-coated tissue culture dishes (ES-qualified, BD Biosciences) with mTeSR1 (STEMCELL Technologies) at 37°C and 5% CO2 in a humidified atmosphere incubator. The iPSCs culture medium was changed daily. The cells were passaged every 3-4 days using Accutase (Stemcell Technologies). The iPS cells conversion to EPS as previous reported 47 (link) . ddPCR ddPCR was performed on a Bio-Rad QX200 Droplet Digital PCR System using ddPCR Supermix for Probes (No dUTP) kit (Bio-Rad, 1863024) according to manufacturer's protocols. The probes were synthesized by Integrated DNA Technologies Inc. as PrimeTime qPCR Probes. The wildtype probes were labeled as 5'HEX/ZEN/3'IBFQ, while the mutant probes were labeled by 5'FAM/ZEN/3'IBFQ. The primer and probe sequences were shown in Supplementary Table 2.
The primer/probe ratio was set as 3.6:1. For each reaction, 0.5 ng of purified 293T cell genome were used. All experiments were performed in three independent replicates, and the positive events were combined for the analysis.
The iPSCs were cultured on Matrigel-coated tissue culture dishes (ES-qualified, BD Biosciences) with mTeSR1 (STEMCELL Technologies) at 37°C and 5% CO2 in a humidified atmosphere incubator. The iPSCs culture medium was changed daily. The cells were passaged every 3-4 days using Accutase (Stemcell Technologies). The iPS cells conversion to EPS as previous reported 47 (link) . ddPCR ddPCR was performed on a Bio-Rad QX200 Droplet Digital PCR System using ddPCR Supermix for Probes (No dUTP) kit (Bio-Rad, 1863024) according to manufacturer's protocols. The probes were synthesized by Integrated DNA Technologies Inc. as PrimeTime qPCR Probes. The wildtype probes were labeled as 5'HEX/ZEN/3'IBFQ, while the mutant probes were labeled by 5'FAM/ZEN/3'IBFQ. The primer and probe sequences were shown in Supplementary Table 2.
The primer/probe ratio was set as 3.6:1. For each reaction, 0.5 ng of purified 293T cell genome were used. All experiments were performed in three independent replicates, and the positive events were combined for the analysis.
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!