HeLa cells were first seeded on coverslips in petri dishes with DMEM culture medium at 37 °C for 24 h. ER-GFP (C10590, Invitrogen) and Actin-RFP (C10583, Invitrogen) plasmids were transfected into cells for 48 h following the protocol from Invitrogen. 500 μM oleic acid (O1383 SIGMA) coupled with BSA in DMEM culture medium was then added to cells for 7 h to induce the formation of lipid droplets. Before imaging, cells were first incubated with 6 μM SYTO60 (S11342, Invitrogen), 120 nM LysoTracker Deep Red (L12492, Invitrogen) and 400 nM Rhodamine 800 (83701 SIGMA) in HBSS simultaneously for 30 min at 37 °C, followed by staining with NucBlue® Live ReadyProbes® Reagent (R37605, Invitrogen) in HBSS for 20 min at 37 °C. Then cells were incubated with ATTO 740-conjugated WGA in HBSS for 30 min at 37 °C, followed by staining with LipidTOX Deep Red (H34477, Invitrogen) in HBSS with a dilution of 1:20 for 30 min at room temperature before imaging.
Actin rfp
Manufactured by Thermo Fisher Scientific
Actin-RFP is a fluorescent protein construct that labels actin, a key structural protein found in the cytoskeleton of cells. It can be used to visualize and study the dynamics of the actin cytoskeleton in living cells.
Automatically generated - may contain errors
Lab products found in correlation
Lipidtox deep red, by Thermo Fisher Scientific (2 mentions)
Rhodamine 800, by Merck Group (2 mentions)
C10590, by Thermo Fisher Scientific (2 mentions)
Syto60, by Thermo Fisher Scientific (2 mentions)
Oleic acid, by Merck Group (2 mentions)
Nucblue live readyprobes reagent, by Thermo Fisher Scientific (2 mentions)
Lysotracker deep red, by Thermo Fisher Scientific (2 mentions)
Fugene 6, by Promega (2 mentions)
Actin gfp, by Thermo Fisher Scientific (2 mentions)
Talin gfp, by Thermo Fisher Scientific (2 mentions)
Tubulin gfp, by Thermo Fisher Scientific (2 mentions)
Standard charge coupled device camera, by Hamamatsu Photonics (2 mentions)
Mitochondria gfp, by Thermo Fisher Scientific (1 mentions)
Golgi gfp bacman 2, by Thermo Fisher Scientific (1 mentions)
Lysosome rfp, by Thermo Fisher Scientific (1 mentions)
5 protocols using actin rfp
1
Multicolor Labeling for Lipid Droplet Imaging
2
Multicolor Cellular Organelle Imaging
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Prior to fixation, the cells were counterstained with CellMask Orange plasma stain (C10045) to identify the cell’s cytoplasmic area. The cells were washed and fixed with an equal amount of warm 4% paraformaldehyde (PF) in phosphate-buffered saline (PBS). To identify intra-cellular structures, the cells were transfected with Golgi GFP BacMan 2.0 (C10592), lysosome-RFP (C10597), mitochondria-GFP (C10600), actin-RFP (C10583), or endoplasmic reticulum-GFP BacMan (C10590) (Invitrogen, Eugene Oregon). Prolong Gold, containing 10 μg/ml DAPI (P36935), was used to mount the slides and stain the nuclei. After the mounting medium dried, slides were sealed and then observed with a combination of dark field and fluorescence microscopy.
3
Multicolor Labeling for Lipid Droplet Imaging
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HeLa cells were first seeded on coverslips in petri dishes with DMEM culture medium at 37 °C for 24 h. ER-GFP (C10590, Invitrogen) and Actin-RFP (C10583, Invitrogen) plasmids were transfected into cells for 48 h following the protocol from Invitrogen. 500 μM oleic acid (O1383 SIGMA) coupled with BSA in DMEM culture medium was then added to cells for 7 h to induce the formation of lipid droplets. Before imaging, cells were first incubated with 6 μM SYTO60 (S11342, Invitrogen), 120 nM LysoTracker Deep Red (L12492, Invitrogen) and 400 nM Rhodamine 800 (83701 SIGMA) in HBSS simultaneously for 30 min at 37 °C, followed by staining with NucBlue® Live ReadyProbes® Reagent (R37605, Invitrogen) in HBSS for 20 min at 37 °C. Then cells were incubated with ATTO 740-conjugated WGA in HBSS for 30 min at 37 °C, followed by staining with LipidTOX Deep Red (H34477, Invitrogen) in HBSS with a dilution of 1:20 for 30 min at room temperature before imaging.
4
Visualizing Cellular Dynamics with Fluorescent Proteins
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To induce the expression of fluorescently tagged proteins chemical transfection or viral transduction were used. In the first case, cells were transfected with Lifeact-GFP using standard chemical transfection reagents (Fugene 6, Promega) and imaged 2 to 3 days after transfection. In the second case, cells were transduced with baculoviruses harboring the constructs Actin-RFP, Actin-GFP, Talin-GFP, and Tubulin-GFP (Life technologies) and imaged 1 to 2 days after transduction. Bright field, phase and fluorescence images were acquired with an inverted epifluorescence fluorescence microscope (AxioObserver.A1, Zeiss) through a 20X air or a 100X oil-immersion objective. Images were captured with a standard charge-coupled device camera (Hamamatsu). For fluorescence measurements, the camera dark noise was subtracted and cells that are high in fluorescence intensity were not included in the analysis. Alignment between fluorescence and AFM images was performed through the alignment of fiducial markers, such as stable cell edges and actin fibers. Data analysis was performed with built-in functions of the software ImageJ.
5
Visualizing Cellular Dynamics with Fluorescent Proteins
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