Sep 06, 2022
Single cell survival assay
- 1University of California, Berkeley
External link: https://doi.org/10.7554/eLife.79208
Protocol Citation: Hanqin Li, Dirk Hockemeyer 2022. Single cell survival assay. protocols.io https://dx.doi.org/10.17504/protocols.io.4r3l2okxxv1y/v1
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 protocol 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 protocol and it's working
Created: July 23, 2022
Last Modified: May 31, 2024
Protocol Integer ID: 67391
Keywords: ASAPCRN
Funders Acknowledgement:
Aligning Science Across Parkinson's
Grant ID: ASAP-000486
Abstract
This protocol describes the experimental procedure used to measure single cell survival rates post nucleofection of human pluripotent stem cells (hPSCs).
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 | |
| 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 |
hPSCs are cultured on MEFs as described in the collection "Thawing, Passaging, and Freezing of hPSCs on MEFS" dx.doi.org/10.17504/protocols.io.b4msqu6e
0.5 million hPSCs are nucleofected as described in “Nucleofection of hPSCs" dx.doi.org/10.17504/protocols.io.b4pcqviw
Seed 1/100, 1/500 of cells post-nucleofection into one well of a 6-well plate containing hPSCs medium with Rock inhibitor. Prepare at least 2 wells for each seeding density as replicates.
hPSC medium
| A | B | |
| DMEM/F12 | 385 ml | |
| Fetal Bovine Serum (FBS) | 75 ml | |
| Knockout Serum Replacement | 25 ml | |
| L-Glutamine (100X) | 5 ml | |
| Penicillin & Streptomycin (100X) | 5 ml | |
| MEM Non-Essential Amino Acids (100X) | 5 ml | |
| 2-Mercaptoethanol (10,000X) | 50 µl | |
| Heat Stable Recombinant Human FGF2 (25 µg/ml)* | 80 µl |
Final volume; 500 mL
*While we prefer Heat Stable Recombinant Human FGF2, we also have used regular FGF2.
L-Glutamine (100x)
| A | B | |
| L-Glutamine, powder | 14.6 g | |
| MilliQ H2O | 500 ml |
2-Mercaptoethanol (10,000X)
| A | B | |
| 2-Mercaptoethanol | 0.78 ml | |
| MilliQ H2O | 9.22 ml |
Heat Stable Recombinant Human FGF2 (25 µg/ml)
| A | B | |
| Heat Stable Recombinant Human FGF2 | 500 µg | |
| 0.1% BSA | 20 ml |
Final volume: 20 ml
hPSCs Medium + Rock inhibitor
| A | B | |
| hPSCs medium | 500 ml | |
| Y-27632 (1,000X) | 500 µl |
Final volume: 500 ml
Y-27632 (1,000X)
| A | B | |
| Y-27632 | 5 mg | |
| DMSO | 1.56 ml |
Change medium at day 3, 6, 8, 10, and then daily until colonies grow to medium size.
Perform Alkaline phosphatase (AP) staining as described in “Pluripotency markers staining” dx.doi.org/10.17504/protocols.io.b4yyqxxw
Image the entire stained well with ChemiDoc (Bio-Rad). Use a white cardboard as background.
Count AP positive colonies, N.
Calculate single cell survival rate by: N x dilution factor/500,000.
E.g. For the wells with seeding density 1/100, the dilution factor is 100.