Sep 06, 2022
Sample preparation for aCGH karyotyping
- 1University of California, Berkeley
External link: https://doi.org/10.7554/eLife.79208
Protocol Citation: Hanqin Li, Dirk Hockemeyer 2022. Sample preparation for aCGH karyotyping. protocols.io https://dx.doi.org/10.17504/protocols.io.kxygxzdrov8j/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: 67380
Keywords: ASAPCRN
Funders Acknowledgement:
Aligning Science Across Parkinson's
Grant ID: ASAP-000486
Abstract
This protocol describes the standard procedure preparing cell pellets from human pluripotent stem cells (hPSCs) cultured on MEFs for outsourced aCGH karyotyping.
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 | |
| Knockout Serum Replacement | Thermo Fisher | 10828-028 | |
| Newborn Calf Serum | Sigma | N4762 | |
| L-Glutamine | Sigma | G8540 | |
| Penicillin & Streptomycin (100X) | Thermo Fisher | 15140163 | |
| MEM Non-Essential Amino Acids (100X) | Thermo Fisher | 11140050 | |
| Collagenase type IV | Thermo Fisher | 17104019 | |
| 40 micron Cell Strainer | Corning | 352340 | |
| DPBS w/o Calcium and magnesium (DPBS) | Corning | MT21031CV |
Collecting hPSCs colonies from MEFs
Collecting hPSCs colonies from MEFs
30m
30m
Use one, almost confluent, 6-well plate of hPSCs to prepare cell pellet
Wash once with DPBS
Add 1 ml of 1 mg/ml collagenase solution into each well to separate hPSC colonies from MEFs. Incubate 00:30:00 at 37 °C .
30m
Collagenase solution
| Collagenase type IV | 10 mg | |
| KSR medium | 10 ml |
1 mg/ml
Final volume: 10 ml
KSR medium
| DMEM/F12 | 385 ml | |
| Knockout Serum Replacement | 100 ml | |
| L-Glutamine (200 mM) | 5 ml | |
| Penicillin & Streptomycin (100X) | 5 ml | |
| MEM Non-Essential Amino Acids (100X) | 5 ml |
Final volume: 500 ml
Add 2 ml Wash Medium to each well
Wash Medium
| DMEM/F12 | 470 ml | |
| Newborn Calf Serum | 25 ml | |
| Penicillin & Streptomycin (100X) | 5 ml |
Final volume: 500 mL
Pipette repeatedly with 5 ml pipette to lift colonies. Be careful not to carry over too many MEFs
Transfer all colonies into a 15 ml tube
Add 7 ml Wash Medium into the 15 ml tube. Pipette with 10 ml Stripette for 5 times to separate MEFs that are attached to hPSCs colonies
Place the 15 ml tubes on a tube rack and gravity settle colonies at Room temperature for 00:05:00 .
5m
Aspirate supernatant and add 10 ml Wash Medium. Invert the tube 3 times to mix.
Gravity settle at Room temperature for 00:05:00 .
5m
Aspirate supernatant and re-suspend colonies in 10 ml Wash Medium
Place a 40 µm cell strainer onto a 50 ml tube
Strain the colony suspension from step 11. Keep the strainer since colonies are trapped on it.
Wash the strainer with 10 ml Wash Medium (x2)
Revert the strainer and place it in a 6-well plate, bottom-up
Apply 5 ml Wash Medium to the bottom of the strainer, twice. This will separate colonies from the strainer and wash them into the 6-well plate.
Collect colonies from the 6-well plate to a new 15 ml tube
Centrifuge the 15 ml tube at 300 x g, 00:05:00
5m
Aspirate most of the medium. Leave 1 ml of medium
Re-suspend colonies in the remaining medium, then transfer them to a 1.8 ml Eppendorf tube
Centrifuge the 1.8 ml tube at 300 x g, 00:05:00
5m
Remove the supernatant and cap the tube
Snap freeze the 1.8 ml tube by placing it in liquid nitrogen for more than 5 min.
Store the snap frozen samples at -80 °C , and ship it to Cell Line Genetics on dry ice