Amaxa human stem cell nucleofection kit

Manufactured by Lonza

Sourced in United States

The Amaxa Human Stem Cell Nucleofection Kit is a laboratory tool designed for the transfection of human stem cells. It provides a method for the efficient delivery of nucleic acids, such as DNA or RNA, into the cells. The kit is intended to facilitate research and experimentation involving human stem cells.

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Lab products found in correlation

Puromycin, by Thermo Fisher Scientific (2 mentions) Accutase, by Thermo Fisher Scientific (2 mentions) Neomycin, by Thermo Fisher Scientific (2 mentions) Tryple express enzyme without phenol red, by Thermo Fisher Scientific (1 mentions)

Spelling variants of this product

Nucleofection (130 citations) Amaxa nucleofection (40 citations) Nucleofection Kit (19 citations) Amaxa nucleofection kit (14 citations) Human nucleofection kit (4 citations) Amaxa kits (2 citations)

3 protocols using amaxa human stem cell nucleofection kit

Generation of Engineered Human iPSCs

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Example 3

Prior to nucleofection, XCL1 iPS cells were maintained and passed using Accutase (Life Tech., NJ) to make sure cells are growing in monolayer. On the day of nucleofection, single cell suspension cells were generated using Accutase followed by inactivation and washes with HBSS. 4-6 ug of each pair of TALENs/ZFNs RNA was used for nucleofection using Amaxa Human Stem Cell Nucleofection Kit (Lonza, NJ). After nucleofection, cells were plated in mTeSR™1 medium with 10 uM Rock inhibitor. After 2-5 days recovery, cells were treated with appropriate antibiotics. Specifically, 2.5 pg/ml Puromycin (Life Tech., NJ) for AAVS1-copGFP, MAP2-nanoluc®-KI and GFAP-Nanoluc®-KI lines and 500 pg/ml Neomycin (Life Tech., NJ) for Chr13-copGFP line. Drug resistant colonies were re-plated at low density for single cell cloning. Colonies growing from single cells were screened by PCRs and sequencing to identify targets with correct donor vector integrations. The verified targets were expanded, stored and characterized for future experiments.

US11530389B2. Methods and compositions for rapid generation of single and multiplexed reporters in cells (2022-12-20). RXCELL INC. [US]. Inventors: Xianmin Zeng [US], Mahendra S. Rao [US].

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Generating genetically engineered iPSCs

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Prior to nucleofection, XCL1 iPSC were maintained and passed using Accutase (Life Tech., NJ) to make sure cells are growing in monolayer. On the day of nucleofection, single cell suspension cells were generated using Accutase followed by inactivation and washes with HBSS. 4–6 μg of each pair of TALENs/ZFNs RNA was used for nucleofection using Amaxa Human Stem Cell Nucleofection Kit (Lonza, NJ). After nucleofection, cells were plated in mTeSR™1 medium with 10 μM Rock inhibitor. After 2–5 days recovery, cells were treated with appropriate antibiotics. Specifically, 2.5 μg/ml Puromycin (Life Tech., NJ) for AAVS1-copGFP, MAP2-nanoluc-KI and GFAP-Nanoluc-KI lines and 500 μg/ml Neomycin (Life Tech., NJ) for Chr13-copGFP line. Drug resistant colonies were re-plated at low density for single cell cloning. Colonies growing from single cells were screened by PCRs and sequencing to identify targets with correct donor vector integrations. The verified targets were expanded, stored and characterized for future experiments.

Pei Y., Sierra G., Sivapatham R., Swistowski A., Rao M.S, & Zeng X. (2015). A platform for rapid generation of single and multiplexed reporters in human iPSC lines. Scientific Reports, 5, 9205.

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Generation of iPSC-Derived Clones

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First, 1.6× 10⁶ of human iPSCs were disassociated and cultured in a 6 cm dish with mTeSR-plus medium for 24 h. When the cells reached 80% confluence, 8 μg of COE plasmid was used for nucleofection with the Amaxa Human Stem Cell Nucleofection Kit (Lonza, MD, USA). On day 3, cells were selected by hygromycin B for 72 h. For the analysis of single cell-derived clones, the cells were disassociated into single cells with TrypLE Express Enzyme without Phenol Red (Thermo Fisher) at day 10 post- nucleofection. They were then seeded onto the Matrigel-coated plates with mTeSR-plus medium for 15 days. Individual colonies were selected, and genotyped PCR was performed to screen for positive clones. The PCR primer’s sequences are shown in Table S1.

Duan N., Tang S., Zeng B., Hu Z., Hu Q., Wu L., Zhou M, & Liang D. (2021). An Episomal CRISPR/Cas12a System for Mediating Efficient Gene Editing. Life, 11(11), 1262.

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