Wild-type or mutant Flag-tagged mDia2 cDNAs (17, 20 (link)) were subcloned into the retroviral pMX vector (Cell Biolabs). pGEM-MIRO1 and cyto-Ruby3-iATPSnFR1.0 (ATP biosensors; ref. 21 (link)) plasmids were purchased from Sino Biological Inc. (#HG15898-G) or Addgene (#102551), respectively. Plasmid vectors and Lipofectamine 2000 (#11668030, Invitrogen) were incubated with Opti-MEM I Reduced Serum Medium (#31985062, Thermo Fisher Scientific) at room temperature for 20 minutes. The mixture was then added to primary human fibroblasts. After 6 hours of incubation, the medium was replaced by a normal culture medium containing 10% FBS. Cells were allowed to recover for 24–48 hours prior to analysis.
Protocol detail
Transient Expression of mDia2 and ATP Biosensors in Human Fibroblasts
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Biological
Biosensors
Cdnas
Cells
Culture medium
Fibroblasts
Human
Lipofectamine 2000
Mdia2
Pgem
Plasmid
Retroviral
Serum
Sino
Vectors
Corresponding Organization : ETH Zurich
Other organizations : University Hospital of Zurich, University of Zurich
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Variable analysis
independent variables
- Wild-type or mutant Flag-tagged mDia2 cDNAs
- Not explicitly mentioned
- Plasmid vectors
- Lipofectamine 2000
- Opti-MEM I Reduced Serum Medium
- Normal culture medium containing 10% FBS
- Positive control: pGEM-MIRO1 and cyto-Ruby3-iATPSnFR1.0 (ATP biosensors) plasmids
- Negative control: Not mentioned
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