Plasmid-reprogramming of human fibroblasts
michela.deleidi, Pascale Baden
Published: 2023-03-18 DOI: 10.17504/protocols.io.eq2lyn8qwvx9/v1
Abstract
This protocol details about plasmid-reprogramming of human fibroblasts.
Attachments
Steps
Nucleofection (Day 0)- with Amaxa Nucleofector I/II
1.
Prepare nucleofection solution:
| A | B |
|---|---|
| Human Dermal Fibroblast Nucleofector solution | 82 µL |
| Supplement | 18 µl |
| Plasmid | 10 µg |
2.
Nucleofect 700.000 fibroblasts (resuspended in the nucleofection solution) with the P-022 program.
3.
Distribute nucleofected fibroblasts on a 6-Well plate, coated with Matrigel.
4.
Culture cells in DMEM + GlutaMAX supplement (Gibco) + 10 % FBS (Gibco) without P/S.
Day 1
5.
Change medium to DMEM + GlutaMAX supplement + 10% FBS + 2ng/mL FGF2 (Peprotech) with 1% P/S (Millipore).
Day 3/4
6.
Change medium to E8 medium + 100micromolar (µM) Sodium Butyrate (Sigma Aldrich 303410-100G) + 0.1 % P/S and change medium every other day.
Day 21-28
7. Note : First colonies should appear around Day 14.
Pick iPS cell colonies.
Note
