Celllight mitochondria gfp

Su9-RFP MEFs, Drp1-knockout Su9-RFP MEFs^{27 (link)}, and HeLa cells (ATCC CCL-2) were grown on glass-bottomed 35-mm culture dishes (Matsunami, Tokyo, Japan) for conventional observation and a μ-Dish 35 mm Grid-500 (Ibidi, Martinsried, Germany) for CLEM in Dulbecco’s modified Eagle’s medium (DMEM) with 10% foetal bovine serum (FBS) in an incubator with 5.0% CO₂ at 37 °C. HeLa cells were transfected with CellLight Mitochondria-GFP or Mitochondria-RFP BacMam2.0 (Thermo Fisher Scientific, Waltham, MA, USA) to visualise mitochondria at 60–70% confluence in complete medium according to the manufacturer’s instructions.

For the staining of mitochondria, lysosomes, and autophagosomes in living cells, CellLight™ 2.0 BacMam technology from Thermo Fisher Scientific (Waltham, MA, USA) was used according to the manufacturer’s protocol. In brief, after 6 weeks of differentiation, an amount of CellLight™ Mitochondria-GFP, Lysosomes-RFP, Premo™ Autophagy Sensor LC3B-GFP, or Premo™ Autophagy Tandem Sensor RFP-GFP-LC3B Kit (all four from Thermo Fischer Scientific) equal to 35 particles per cell was diluted in culture medium, added to each well, and incubated for 24 h. Cells were then transformed to the imaging chambers containing EXS-HEPES buffer (151 mM NaCl, 1.25 mM NaH₂PO₄, 10 mM HEPES, 2.5 mM KCl, 2 mM CaCl₂, 1 mM MgCl₂, 10mM Glucose, pH 7.4), and live cell imaging was performed with a Zeiss AxioObserver microscope (Zeiss, Hamburg, Germany). Cells were kept at 37 °C during microscopy. Four to five videos with 300 frames and a frame rate of 4 frames per second were recorded of four independent experiments per cell line and condition.

Fibroblasts were split at 5000 cells/500 µl of FM, immediately infected with 2 µl of CellLight Mitochondria-GFP, BacMam 2.0 (ThermoFisher), and plated in Lab-Tek II Chambered Coverglass w/cover #1.5 4-well Borosilicate Sterile plates (ThermoFisher). After 72 h of incubation at 37 ^oC in 5% CO_2, the cells were placed in a temperature and CO₂-controlled chamber (Tokai Hit) for live imaging. Images were acquired on a Nikon A1 confocal microscope with a ×40 oil objective lens (1.3 N.A.) and excited with a 543 nm-wavelength HeNe laser. NIS-Elements Software (Nikon Instruments) was used to acquire images of individual cells at 1024 × 1024 pixel resolution. Approximately 20 images were taken for each cell line and the cell identity was masked from the observer during analysis. Mitochondrial morphology (including form factor and aspect ratio) were determined using a mitochondrial morphology macro⁷⁷ in Fiji.
For analysis of mitochondrial morphology in iPSCs, cultures were grown for 4−5 d after passaging and treated with 200 nM tetramethylrhodamine, ethyl ester (TMRE) directly in E8 media for 15 min at 37 °C. Colonies were washed once with PBS before replacing the media. Imaging was accomplished using a Nikon TiE/B inverted fluorescence microscope with an Andor Zyla 4.2p sCOMS camera, and mitochondrial morphology assessed as above.

After 48 h of fibroblast culture, the culture medium from a 25 mL culture flask was collected and extracellular vesicles were purified according to the method described in [15 (link)]. HL-1 cells were transfected using CellLight™ Mitochondria-GFP, BacMam 2.0 (ThermoFisher Scientific, Waltham, MA, USA) and incubated with the vesicles for 24 h. Alternatively, the cells were preincubated with 10 µM forskolin (Merck, Darmstadt, Germany) before application of EVs or treated with 2 µM PKA inhibitor (KT5720, Merck, Darmstadt, Germany) for 24 h. The velocity of at least 144 mitochondria was measured for each treatment. The experiment was performed in duplicate.
To test the effect of PKA inhibitor on the ability of nocodazole to destabilize microtubules, HL-1 cells were pretreated with a 2 µM PKA inhibitor for 15 min and then incubated with 0.1 µM nocodazole (Merck, Darmstadt, Germany) for 1 h. Cells were fixed, and microtubules were stained as described in the Immunofluorescence subsection.

All solvents were purchased from commercial sources and used as received unless stated otherwise. Hoechst 33342, Invitrogen™ Live Cell Imaging Solution, CellMask™ Green plasma membrane stain, CellLight™ Mitochondria-GFP with BacMam 2.0 were purchased from Thermo Fisher Scientific. Phorbol-12-myristate-13-acetate (PMA) was ordered from Sigma-Aldrich.

PDAC053T cells were seeded at 200000 cells per well 24 h prior to the experiment in 1 μL of medium. Cells were then transduced with CellLight Lysosomes-GFP, BacMam 2.0 (Thermo Fisher Scientific, C10596), CellLight ER-GFP, BacMam 2.0 (Thermo Fisher Scientific, C10590), CellLight Mitochondria-GFP, BacMam 2.0 (Thermo Fisher Scientific, C10600) according to the manufacturer’s procedure. In brief, 70 μL of BacMam reagent was added to the medium and mixed. Cells were incubated for 16 h.