Improve Research Reproducibility A Bio-protocol resource
Concise Method
tooltip

Transfection of HEK293T cells for ICC

FT Franziska R. Traube
DÖ Dilara Özdemir
HS Hanife Sahin
CS Constanze Scheel
AG Andrea F. Glück
AG Anna S. Geserich
SO Sabine Oganesian
SK Sarantos Kostidis
KI Katharina Iwan
RR René Rahimoff
GG Grazia Giorgio
MM Markus Müller
FS Fabio Spada
MB Martin Biel
JC Jürgen Cox
MG Martin Giera
SM Stylianos Michalakis
TC Thomas Carell
request Request a Protocol
ask Ask a question
Favorite

The transfection was performed in 15 µ—slide eight-well plates (ibidi 80826). A total of 2–3 × 104 cells were seeded per well in 200 µL of medium. Twenty-four hours after seeding, the cells were transfected using 150 ng of DNA per expression plasmid, 0.5 µL of jetPRIME, and 15 µL of jetPRIME buffer. Twenty-four hours after transfection, the cells were washed once with PBS supplemented with MgCl2 and CaCl2 (PBS+, Dulbecco’s phosphate buffered saline, Sigma-Aldrich D8662) and immunofluorescence staining was performed.

In case of the bicistronic vector construct, cells were transfected with 200 ng plasmid DNA. 4 h after transfection, expression was induced by doxycycline (1 µg/mL final concentration) and 24 h after induction, cells were washed once with PBS+ and ICC was performed.

Copyright and License information: The Author(s) ©2021
Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

Do you have any questions about this protocol?

Post your question to gather feedback from the community. We will also invite the authors of this article to respond.

post Post a Question
0 Q&A