Abstract
This protocol provides detailed instructions for transient expression of PIP3/PI(3,4)P2-selective PH domain-based reporters for subsequent live-cell imaging by total internal reflection fluorescence microscopy. Instructions are also provided for execution of perturbation experiments using growth factors or inhibitors that act through the PI3K signalling pathway. The protocol was originally developed with advice from Dr York Posor (Vanhaesebroeck Lab, University College London), Dr James Burchfield (University of Sydney) and Dr Alison Kearney (David James Lab, University of Sydney).
The protocol is routinely used in our lab for experiments with HeLa cells. It has also been used with mouse embryonic fibroblasts and lung adenocarcinoma A549 cells, however the complete medium (CM), seeding densities and transfection conditions differ for each cell line.
We culture our cells without antibiotics except during live-cell imaging as indicated.
For subsequent data analysis, please refer to the pipeline provided on the Open Science Framework under doi: 10.17605/OSF.IO/4F69N.
The protocol is routinely used in our lab for experiments with HeLa cells. It has also been used with mouse embryonic fibroblasts and lung adenocarcinoma A549 cells, however the complete medium (CM), seeding densities and transfection conditions differ for each cell line.
We culture our cells without antibiotics except during live-cell imaging as indicated.
For subsequent data analysis, please refer to the pipeline provided on the Open Science Framework under doi: 10.17605/OSF.IO/4F69N.
| Original language | English |
|---|---|
| Type | Protocol |
| Publisher | protocols.io |
| DOIs | |
| Publication status | Published - 21 Dec 2023 |