Sep 06, 2022
Standard operating procedure for the isolation of genetically engineered hPSCs clones in a high-throughput way
- Hanqin Li1,
- Yogendra Verma1,
- Dirk Hockemeyer1,
- Frank Soldner2
- 1University of California, Berkeley;
- 2Albert Einstein College of Medicine
External link: https://doi.org/10.7554/eLife.79208
Collection Citation: Hanqin Li, Yogendra Verma, Dirk Hockemeyer, Frank Soldner 2022. Standard operating procedure for the isolation of genetically engineered hPSCs clones in a high-throughput way. protocols.io https://dx.doi.org/10.17504/protocols.io.b4mmqu46
Manuscript citation:
Hanqin Li, Oriol Busquets, Yogendra Verma, Khaja Mohieddin Syed, Nitzan Kutnowski, Gabriella R Pangilinan, Luke A Gilbert, Helen S Bateup, Donald C Rio, Dirk Hockemeyer, Frank Soldner (2022) Highly efficient generation of isogenic pluripotent stem cell models using prime editing eLife 11:e79208
License: This is an open access collection distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this collection and it's working
Created: February 02, 2022
Last Modified: December 13, 2024
Collection Integer ID: 57741
Keywords: ASAPCRN, nucleofection of hpsc, cultured hpsc, isolating single human pluripotent stem cell, single human pluripotent stem cell, term hpsc, hpsc, human pluripotent stem cell, confirmed hpsc, subcloning of genotype, standard operating procedure for the isolation, clone, genotype, subcloning, described procedure, hesc, biorad, standard operating procedure, nucleofection, hipsc
Funders Acknowledgements:
Aligning Science Across Parkinson's
Grant ID: ASAP-000486
Abstract
This collection describes a standard procedure for isolating single human pluripotent stem cell (hPSC) clones in a high-throughput way. This collection follows nucleofection of hPSCs as described in detail in the collection "Nucleofection (Amaxa) and electroporation (Biorad) of hPSCs;" dx.doi.org/10.17504/protocols.io.b4qnqvve
Collection overview
Seeding nucleofected hPSCs in 96-well plates using limited dilution
Duplicating 96-well plate-cultured hPSCs clones
Subcloning of genotype-confirmed hPSCs clones
General notes
1. Throughout these protocols, the term hPSC is used to collectively refer to both hiPSCs and hESCs. All described procedures have been tested and work equally well for hiPSCs and hESCs.
Materials
| A | B | C | |
| Item | Vendor | Catalog # | |
| DMEM/F12 | Thermo Fisher | 11320082 | |
| DPBS w/o Calcium and magnesium (DPBS) | Corning | MT21031CV | |
| Fetal Bovine Serum (FBS) | Corning | 35-011-CV | |
| Knockout Serum Replacement | Thermo Fisher | 10828-028 | |
| L-Glutamine | Sigma | G8540 | |
| Penicillin & Streptomycin (100X) | Thermo Fisher | 15140163 | |
| MEM Non-Essential Amino Acids (100X) | Thermo Fisher | 11140050 | |
| Heat Stable Recombinant Human FGF2 * | Thermo Fisher | PHG0360 | |
| Y-27632 | Chemdea | CD0141 | |
| 2-Mercaptoethanol | Sigma | M3148 | |
| 0.25% Trypsin with EDTA (Trypisin) | Thermo Fisher | 25200114 | |
| DMSO | Fisher Scientific | BP231-100 | |
| Proteinase K | Sigma | P6556 |
*While we prefer Heat Stable Recombinant Human FGF2, we also have used regular FGF2
Troubleshooting