Sample preparation for aCGH karyotyping
Hanqin Li, Dirk Hockemeyer
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:
- 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.
Steps
Collecting hPSCs colonies from MEFs
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 0h 30m 0s at 37°C.
Collagenase solution
| A | B |
|---|---|
| Collagenase type IV | 10 mg |
| KSR medium | 10 ml |
1 mg/mlFinal volume: 10 ml
KSR medium
| A | B |
|---|---|
| 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
| A | B |
|---|---|
| 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 0h 5m 0s.
Aspirate supernatant and add 10 ml Wash Medium. Invert the tube 3 times to mix.
Gravity settle at Room temperature for 0h 5m 0s.
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 300x g
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 300x g
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
