Hematopoietic progenitor cells were electroporated with CRISPR-Cas9 plasmids, as previously described, using Amaxa Nucleofector I (Lonza) and following the manufacturer’s protocols. Cells were electroporated in cuvettes (electroporation volume of 100 μL) using a human CD34+ cells kit (Lonza) and the U08 electroporation program. The total amount of plasmid used was 11.26 μg; hence, when two plasmids were electroporated at the same time, 5.63 μg of each was added to the mix. After electroporation, cells were cultured on a 24-well plate (Falcon) at 2 mL of final volume. In the case of the experiments with selection based on ZsGreen reporter expression, sorting was performed 24 h after electroporation. Aggregates were eliminated before sorting using a 40-μm cell strainer (Falcon).
Human cd34 cells kit
Manufactured by Lonza
The Human CD34+ Cells Kit is a laboratory product that contains human CD34+ cells. CD34+ cells are a type of stem cell that can differentiate into various blood and immune cell types. The kit provides a source of these cells for research and experimental purposes.
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2 protocols using human cd34 cells kit
1
Electroporation of Hematopoietic Progenitors
2
CRISPR/Cas9 Electroporation of CD34+ Cells
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Hematopoietic progenitor cells were electroporated with CRISPR/Cas9 plasmids as previously described, using Amaxa Nucleofector I (Lonza) and following manufacturer´s protocols. Cells were electroporated in cuvettes (electroporation volume of 100 µL) using Human CD34 + Cells kit (Lonza) and U08 electroporation program. Total amount of plasmid used was 11.26 µg, hence, when two plasmids were electroporated at the same time, 5.63 µg of each were added to the mix. After electroporation, cells were cultured on 24-well plate (FALCON) at 2 mL of final volume. In the case of the experiments with selection based on ZsGreen reporter expression, sorting was performed 10 24 hours after electroporation. Aggregates were eliminated before sorting using 40 µm Cell Strainer (FALCON).
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