Mar 30, 2023

Public workspacePlasmid-reprogramming of human fibroblasts

DOI
dx.doi.org/10.17504/protocols.io.5qpvor45bv4o/v1
  • 1German Center for Neurodegenerative Diseases (DZNE), Tübingen, 72076 Germany
Federico Bertoli
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DOI: dx.doi.org/10.17504/protocols.io.5qpvor45bv4o/v1
Protocol Citation: Pascale Baden, michela.deleidi 2023. Plasmid-reprogramming of human fibroblasts. protocols.io https://dx.doi.org/10.17504/protocols.io.5qpvor45bv4o/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: March 28, 2023
Last Modified: May 31, 2024
Protocol Integer ID: 79540
Keywords: Nucleofection, Plasmid-reprogramming, human fibroblasts, ASAPCRN
Abstract
This protocol details about plasmid-reprogramming of human fibroblasts.
Attachments
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404-873.docx
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Guidelines
Adapted from Okita et al PMID: 21460823.
Materials
Materials

  • DMEM
  • GlutaMAX supplement (Gibco)
  • FBS (Gibco)
  • FGF2 (Peprotech)
  • Nucleofection Kit for Amaxa Nucleofector: Normal Human Dermal Fibroblasts
ReagentHuman Dermal Fibroblast Nucleofector™ KitLonzaCatalog #VPD-1001
ReagentSodium butyrateSigma AldrichCatalog #303410

Plasmids (from Addgene):
ReagentpCXLE-hOCT3/4addgeneCatalog #27076 ReagentpCXLE-hSKaddgeneCatalog #27078 ReagentpCXLE-hULaddgeneCatalog #27080


Nucleofection (Day 0)- with Amaxa Nucleofector I/II
Nucleofection (Day 0)- with Amaxa Nucleofector I/II
Prepare nucleofection solution:
AB
Human Dermal Fibroblast Nucleofector solution82 µL 
Supplement18 µl 
Plasmid10 µg 
Nucleofect 700.000 fibroblasts (resuspended in the nucleofection solution) with the P-022 program.
Distribute nucleofected fibroblasts on a 6-Well plate, coated with Matrigel.
Culture cells in DMEM + GlutaMAX supplement (Gibco) + 10 % FBS (Gibco) without P/S.
Day 1
Day 1
Change medium to DMEM + GlutaMAX supplement + 10% FBS + Amount2 undetermined FGF2 (Peprotech) with 1% P/S (Millipore).

Day 3/4
Day 3/4
Change medium to E8 medium + Concentration100 micromolar (µM) Sodium Butyrate (Sigma Aldrich 303410-100G) + 0.1 % P/S and change medium every other day.

Day 21-28
Day 21-28
Pick iPS cell colonies.
Note
Note: First colonies should appear around Day 14.