Preparing feeder-free hPSCs for nucleofection

Abstract

This protocol describes the standard procedure preparing feeder-free human pluripotent stem cells (hPSCs) for nucleofection.

General notes

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.
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:
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

Cells are ready for nucleofection when the culture reaches 70-80% confluency

For a detailed protocol on growing feeder-free hPSCs, refer to the collection "Feeder-free culturing of hPSCs;" dx.doi.org/10.17504/protocols.io.b4mcqu2w

Coat 6-well plates with VTN/Matrigel/Geltrex as depicted in the collection “Feeder-free culturing of hPSCs," dx.doi.org/10.17504/protocols.io.b4mcqu2w

Wash hPSCs with DPBS

Use 1 ml Accutase/well of a 6-well plate.

Incubate 0h 5m 0s 37°C

Add 2 ml DMEM/F12 to each well.

Collect all cells into 15 ml conical tube.

Add 7 ml DMEM/F12.

Centrifuge at 200-300x g

10.

Aspirate supernatant

11.

Resuspend cell pellet in 1 ml DMEM/F12, triturate to single cells using P1000 tips

12.

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

13.

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.

14.

Mix the cell suspension in the conical tube, take 500,000 cells per nucleofection reaction and transfer to a new conical tube

15.

Centrifuge at 200-300x g

16.

Aspirate supernatant

17.

Re-suspend cell pellet in 10 ml DPBS

18.

Centrifuge at 200-300x g

19.

Aspirate supernatant as much as possible, to minimize the interference to the nucleofection buffer system.