Cell Navigator® F-Actin Labeling Kit *Red Fluorescence*
Our Cell Navigator® fluorescence imaging kits are a set of fluorescence imaging tools for labeling sub-cellular organelles such as membranes, lysosomes, mitochondria and nuclei etc. The selective labeling of live cell compartments provides a powerful method for studying cellular events in a spatial and temporal context. This particular kit is designed to label F-actins of fixed cells in red fluorescence. The kit uses a red fluorescent phalloidin conjugate that is selectively bound to F-actins. This red fluorescent phalloidin conjugate is a high-affinity probe for F-actins. Used at nanomolar concentrations, phallotoxins are convenient probes for labeling, identifying and quantitating F-actins in formaldehyde-fixed and permeabilized tissue sections, cell cultures or cell-free experiments. The labeling protocol is robust, requiring minimal hands-on time. The kit provides all the essential components with an optimized staining protocol.
Example protocol
AT A GLANCE
Protocol summary
- Prepare samples (microplate wells)
- Remove the liquid from the plate
- Add 100 µL/well of iFluor™ 594-Phalloidin working solution
- Stain the cells at RT for 15 to 60 minutes
- Wash the cells
- Examine the specimen under fluorescence microscope at Ex/Em = 594/610 nm (Texas Red channel)
Important notes
Thaw all the components at room temperature before starting the experiment.
PREPARATION OF WORKING SOLUTION
Add 10 μL of iFluor™ 594-Phalloidin (Component A) to 10 mL of Labeling Buffer (Component B) to make 1X iFluor™ 594-Phalloidin working solution. Protect from light. Note: Different cell types might be stained differently. The concentration of iFluor™ 594-Phalloidin working solution should be prepared accordingly.
For guidelines on cell sample preparation, please visit
https://www.aatbio.com/resources/guides/cell-sample-preparation.html
SAMPLE EXPERIMENTAL PROTOCOL
- Perform formaldehyde fixation. Incubate the cells with 3.0 – 4.0% formaldehyde in PBS at room temperature for 10 – 30 minutes. Note: Avoid any methanol containing fixatives since methanol can disrupt actin during the fixation process. The preferred fixative is methanol-free formaldehyde.
- Rinse the fixed cells 2 – 3 times in PBS.
- Optional: Add 0.1% Triton X-100 in PBS into fixed cells for 3 to 5 minutes to increase permeability. Rinse the cells 2 – 3 times in PBS.
- Add 100 µL/well (96-well plate) of iFluor™ 594-Phalloidin working solution into the fixed cells.
- Stain the cells at room temperature for 15 to 60 minutes.
- Rinse cells gently with PBS 2 to 3 times to remove excess dye before plate sealing.
- Image cells using a fluorescence microscope with Texas Red channel (Ex/Em = 594/610 nm).
Spectrum
Open in Advanced Spectrum Viewer
Product family
Name | Excitation (nm) | Emission (nm) | Extinction coefficient (cm -1 M -1) | Quantum yield | Correction Factor (260 nm) | Correction Factor (280 nm) |
Cell Navigator® F-Actin Labeling Kit *Blue Fluorescence* | 345 | 450 | 200001 | 0.951 | 0.83 | 0.23 |
Cell Navigator® F-Actin Labeling Kit *Green Fluorescence* | 491 | 516 | 750001 | 0.91 | 0.21 | 0.11 |
Cell Navigator® F-Actin Labeling Kit *Orange Fluorescence* | 541 | 557 | 1000001 | 0.671 | 0.25 | 0.15 |
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References
View all 42 references: Citation Explorer
Velocity distributions of single F-actin trajectories from a fluorescence image series using trajectory reconstruction and optical flow mapping
Authors: von Wegner F, Ober T, Weber C, Schurmann S, Winter R, Friedrich O, Fink RH, Vogel M.
Journal: J Biomed Opt (2008): 54018
Authors: von Wegner F, Ober T, Weber C, Schurmann S, Winter R, Friedrich O, Fink RH, Vogel M.
Journal: J Biomed Opt (2008): 54018
Visualization of F-actin and G-actin equilibrium using fluorescence resonance energy transfer (FRET) in cultured cells and neurons in slices
Authors: Okamoto K, Hayashi Y.
Journal: Nat Protoc (2006): 911
Authors: Okamoto K, Hayashi Y.
Journal: Nat Protoc (2006): 911
The effect of F-actin on the relay helix position of myosin II, as revealed by tryptophan fluorescence, and its implications for mechanochemical coupling
Authors: Conibear PB, Malnasi-Csizmadia A, Bagshaw CR.
Journal: Biochemistry (2004): 15404
Authors: Conibear PB, Malnasi-Csizmadia A, Bagshaw CR.
Journal: Biochemistry (2004): 15404
Analysis of models of F-actin using fluorescence resonance energy transfer spectroscopy
Authors: Moens PD, dos Remedios CG.
Journal: Results Probl Cell Differ (2001): 59
Authors: Moens PD, dos Remedios CG.
Journal: Results Probl Cell Differ (2001): 59
Fluorescence studies of the carboxyl-terminal domain of smooth muscle calponin effects of F-actin and salts
Authors: Bartegi A, Roustan C, Kassab R, Fattoum A.
Journal: Eur J Biochem (1999): 335
Authors: Bartegi A, Roustan C, Kassab R, Fattoum A.
Journal: Eur J Biochem (1999): 335
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