TITLE: Sample preparation for aCGH karyotyping
AUTHORS: Hanqin  Li, Dirk Hockemeyer

[DESCRIPTION]
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.

[STEPS]
SECTION: Collecting hPSCs colonies from MEFs
1. Use one, almost confluent, 6-well plate of hPSCs to prepare cell pellet
SECTION: Collecting hPSCs colonies from MEFs
2. Wash once with DPBS
SECTION: Collecting hPSCs colonies from MEFs
3. Add 1 ml of 1 mg/ml collagenase solution into each well to separate hPSC colonies from MEFs. Incubate 30 min at 37 °C.

SECTION: Collecting hPSCs colonies from MEFs
4. Add 2 ml Wash Medium to each well
SECTION: Collecting hPSCs colonies from MEFs
5. Pipette repeatedly with 5 ml pipette to lift colonies. Be careful not to carry over too many MEFs
SECTION: Collecting hPSCs colonies from MEFs
6. Transfer all colonies into a 15 ml tube
SECTION: Collecting hPSCs colonies from MEFs
7. 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
SECTION: Collecting hPSCs colonies from MEFs
8. Place the 15 ml tubes on a tube rack and gravity settle colonies at Room temperature for 5 min. 

SECTION: Collecting hPSCs colonies from MEFs
9. Aspirate supernatant and add 10 ml Wash Medium. Invert the tube 3 times to mix.
SECTION: Collecting hPSCs colonies from MEFs
10. Gravity settle at Room temperature for 5 min.

SECTION: Collecting hPSCs colonies from MEFs
11. Aspirate supernatant and re-suspend colonies in 10 ml Wash Medium
SECTION: Collecting hPSCs colonies from MEFs
12. Place a 40 µm cell strainer onto a 50 ml tube
SECTION: Collecting hPSCs colonies from MEFs
13. Strain the colony suspension from step 11. Keep the strainer since colonies are trapped on it. 
SECTION: Collecting hPSCs colonies from MEFs
14. Wash the strainer with 10 ml Wash Medium (x2)
SECTION: Collecting hPSCs colonies from MEFs
15. Revert the strainer and place it in a 6-well plate, bottom-up
SECTION: Collecting hPSCs colonies from MEFs
16. 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. 
SECTION: Collecting hPSCs colonies from MEFs
17. Collect colonies from the 6-well plate to a new 15 ml tube
SECTION: Collecting hPSCs colonies from MEFs
18. Centrifuge the 15 ml tube at 300 x g, 5 min 

SECTION: Collecting hPSCs colonies from MEFs
19. Aspirate most of the medium. Leave 1 ml of medium
SECTION: Collecting hPSCs colonies from MEFs
20. Re-suspend colonies in the remaining medium, then transfer them to a 1.8 ml Eppendorf tube
SECTION: Collecting hPSCs colonies from MEFs
21. Centrifuge the 1.8 ml tube at 300 x g, 5 min

SECTION: Collecting hPSCs colonies from MEFs
22. Remove the supernatant and cap the tube
SECTION: Collecting hPSCs colonies from MEFs
23. Snap freeze the 1.8 ml tube by placing it in liquid nitrogen for more than 5 min. 
SECTION: Collecting hPSCs colonies from MEFs
24. Store the snap frozen samples at -80 °C, and ship it to Cell Line Genetics on dry ice

SECTION: Collecting hPSCs colonies from MEFs
4.1. Wash Medium
 
     DMEM/F12      470 ml        Newborn Calf Serum      25 ml        Penicillin & Streptomycin (100X)      5 ml   
 
SECTION: Collecting hPSCs colonies from MEFs
3.1. Collagenase solution 
     Collagenase type IV      10 mg        KSR medium      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