TITLE: Preparing MEF-cultured hPSCs for nucleofection
AUTHORS: Hanqin  Li, Oriol Busquets, Steven Poser, Dirk Hockemeyer, Frank Soldner

[DESCRIPTION]
This protocol describes the procedure or preparing MEF-cultured human pluripotent stem cells (hPSCs) for the delivery of plasmids, mRNA or ribonucleoprotein (RNP) using nucleofection.

General notes
1. Throughout this protocol, 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.
2. This protocol is to prepare cells for protocol nucleofection of hPSCs. Before starting, familiarize yourself with the protocol and the required preparations. A detailed protocol on maintaining MEF-cultured hPSCs can be found in the collection "Maintenance and inactivation of mouse embryonic fibroblasts (MEFs) as feeder cells for human pluripotent stem cell culture;" doi:
3. Detailed protocols for preparing plasmids, RNA, and RNP for nucleofection can be found in the collection "Nucleofection (Amaxa) and electroporation (Biorad) of hPSCs." A link to this collection can be found in the title section of this protocol, located above



[STEPS]
1. When MEF-cultured hPSCs reach 50% confluency, change medium to hPSCs medium + Rock inhibitor, preparing for nucleofection the next day. For each 20 µl nucleofection reaction, prepare half to 1 well of cells.

A detailed protocol on maintaining MEF-cultured hPSCs can be found in the collection "Maintenance and inactivation of mouse embryonic fibroblasts (MEFs) as feeder cells for human pluripotent stem cell culture;" dx.doi.org/10.17504/protocols.io.b4pbqvin

2. Prepare feeder plate at least 1 day earlier as depicted in the collection "Maintenance and inactivation of mouse embryonic fibroblasts (MEFs) as feeder cell for human pluripotent stem cell culture," dx.doi.org/10.17504/protocols.io.b4pbqvin
3. Wash MEF-cultured hPSCs with DPBS 
4. Add 1 ml Dissociating Solution to each well
5. Incubate 30 min37 °C . Watch for edge curling of the colonies as indication collagenase incubation is complete.


6. Add 2 ml DMEM/F12 to each well
7. Pipette repeatedly with 5 ml pipette to lift colonies, careful not to carry over too many MEFs.
8. Collect into 15 ml conical tube.
9. Add 7 ml DMEM/F12.
10. Centrifuge at 200-300 x g, 5 min 

11. Aspirate supernatant 
12. Re-suspend cell pellet in 1 ml pre-warmed Accutase
13. Incubate 37 °C 5 min 


14. Add 9 ml DMEM/F12, invert to mix
15. Centrifuge at  200-300 x g, 5 min 

16. Aspirate supernatant
17. Resuspend cell pellet in 1 ml DMEM/F12, triturate to single cells using P1000 tips


18. Take two 10 µl sets of the cell suspension.. Mix each set with 10 µl trypan blue dye which comes with the Countess™ Cell Counting Chamber Slides


19. Count cells with Countess automated cell counter or hemocytometer, average the counts from the two sets. Continue with re-suspending the cell pellet in 20 ml nucleofection solution as described in the protocol "Nucleofection of hPSCs" (Step 2)

The protocol "Nucleofection of hPSCs" can be found in the collection "Nucleofection (Amaxa) and electroporation (Biorad) of hPSCs." A link to this collection can be found in the title section of this protocol, located above
20. Mix the cell suspension in the conical tube, take 500,000 cells per nucleofection reaction and transfer to a new conical tube
21. Centrifuge at 200-300 x g, 5 min 

22. Aspirate supernatant
23. Resuspend cell pellet in 10 ml DPBS 
24. Centrifuge at 200-300 x g, 5 min 

25. Aspirate supernatant as much as possible, to minimize the interference to the nucleofection buffer system.
1.1.  hPSCs medium
     DMEM/F12      385ml        Fetal Bovine Serum (FBS)      75ml        Knockout Serum Replacement      25ml        L-Glutamine (100X)      5ml        Penicillin & Streptomycin (100X)      5ml        MEM Non-Essential Amino Acids (100X)      5ml        2-Mercaptoethanol (10,000X)      50µl        Heat Stable Recombinant Human FGF2 (25µg/ml)      80µl   
 

L-Glutamine (100X)
     L-Glutamine,   powder      14.6 g        MilliQ H2O      500 ml   
 
2-Mercaptoethanol (10,000X)
     2-Mercaptoethanol      0.78 ml        MilliQ H2O      9.22 ml   
 

Heat Stable Recombinant Human FGF2 (25µg/ml)
     Heat Stable Recombinant Human FGF2      500 ug        0.1% BSA      20 ml   
 
Y-27632 (1,000X)
     Y-27632      5 mg        DMSO      1.56ml   
 
hPSCs medium + Rock inhibitor, 500ml
     hESCs medium      500ml        Y-27632 (1,000X)      500  µl   


4.3.  Dissociating solution, 10ml
     Collagenase solution   (10mg/ml)      1ml        Dispase (1U/ml)      5ml        DMEM/F12      4ml   
 
4.1.  Collagenase solution (10mg/ml)
     Collagenase type IV      100 mg        KSR medium      10ml   
 
4.2.  KSR medium
     DMEM/F12      385ml        Knockout Serum Replacement      100ml        L-Glutamine (200 mM)      5ml        Penicillin & Streptomycin (100X)      5ml        MEM Non-Essential Amino Acids (100X)      5ml