About 2.5 × 104 cells were seeded in chamber slides (Ibidi). After 24 h of incubation, the cells were transfected with pEGFP-C1 LifeAct-EGFP (Addgene 58470) using the K2® Transfection Reagent from Biontex according to the manufacturer’s instructions. After incubation for 24 h, time series were taken every 50 s for 17 frames by fluorescence microscopy at 100-fold magnification using the IXplore Live microscope imaging system from Olympus. Three filopodia per cell were measured by analysing its velocity (distance/time; µm/sec) using the Fiji software.
Pegfp c1 lifeact egfp
Manufactured by Addgene
PEGFP-C1 LifeAct-EGFP is a plasmid that encodes a fusion protein of the actin-binding protein LifeAct and the enhanced green fluorescent protein (EGFP). The LifeAct-EGFP fusion protein can be used to visualize the actin cytoskeleton in live cells.
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4 protocols using pegfp c1 lifeact egfp
1
Microscopic Analysis of Filopodia Dynamics
2
EGFP-ITPKA Transfection and Imaging
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A total of 2.5 × 104 cells were seeded in chamber slides (Ibid). After 16 h of incubation, the cells were transfected with EGFP-ITPKA [4 (link)] or pEGFP-C1 LifeAct EGFP (Addgene 58470) using the K2® Transfection Reagent from Biontex according to the manufacturer’s instructions. After further incubation for 24 h, the cells were optionally fixed with 4% paraformaldehyde/4% sucrose, stained with Alexa568-coupled phalloidin, and analyzed by fluorescence microscopy using the IXplore Live microscope imaging system from Olympus.
3
Quantifying Cell-Cell Fusion Dynamics
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CHO cells were seeded at 50,000 cells in 48-well dish 24 hours before transfection. Cells were transfected with 200 mg of pCAGSS-RBP-HA (of MeV/CDV/MBaMV), 200 mg of pCAGGS-F-AU1(of MeV/CDV/MBaMV), pCAGGS-Igk-HA-CD150 (20 ng human, 5 ng dog, or 20 ng bat), and 50 mg of pEGFP-C1 Lifeact-EGFP (purchased from Addgene) with 2.5 ml of polyethylenimine max (polysciences). At 36 hours post transfection, cells were imaged with a Celigo imaging cytometer (Nexcelom) with the GFP channel, and pictures were exported at the resolution of 5 micrometer / pixel. The GFP-positive foci (single cell or syncytia) were analyzed by ImageJ (developed by NIH), creating the profile of individual GFP-positive foci with size information.
For the evaluation of syncytia size, we first filtered the GFP-positive foci with the size of >= 10 pixel2, which is the median size of GFP area in the well of MeV-F plus LifeactGFP transfection to exclude non-specific background noise. Then we calculated the frequency of syncytia which is defined as the GFP counts of >= 100 pixel2 (10 times of median size of single cells) / total GFP counts of >= 10 pixel2.
For the evaluation of syncytia size, we first filtered the GFP-positive foci with the size of >= 10 pixel2, which is the median size of GFP area in the well of MeV-F plus LifeactGFP transfection to exclude non-specific background noise. Then we calculated the frequency of syncytia which is defined as the GFP counts of >= 100 pixel2 (10 times of median size of single cells) / total GFP counts of >= 10 pixel2.
4
Stable Cell Lines Expression Optimization
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Stable nontargeted transfectants were generated and expanded from single-cell clones in exactly the same way as targeted transfectants, except for the electrical pulse, which was delivered at 250 V, 950 μF. Correct expressions were verified by Western blotting and flow cytometry. To generate INPP5BDegron/Degron cells stably expressing LifeAct-EGFP and EGFP-Tubby, cells were transfected with pcDNA-Hygro-LifeAct and pcDNA-Hygro-Tubby, respectively. Stable clones were selected by Hygromycin B at a final concentration of 2 mg/ml in culture medium for 5 d, before FACS-sorting expressing cells based on the EGFP signal. To generate the expressing plasmids, the insert in pcDNA3.1-Hygro-EYFP-H148Q plasmid (#25873; Addgene) was replaced with the coding sequences for EGFP-Tubby or LifeAct-EGFP to yield pcDNA-Hygro-Tubby or pcDNA-Hygro-LifeAct, respectively. The coding sequences for EGFP-Tubby and LifeAct-EGFP were PCR-amplified from pEGFP-C1-Tubby (Martin Lowe laboratory, University of Manchester) and pEGFP-C1-LifeAct-EGFP (#58470; Addgene) using primers PCR-EGFP-Tubby-F/R and PCR-Lifeact-EGFP-F/R, respectively.
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