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Cell Navigator® F-Actin Labeling Kit *Red Fluorescence*

Fluorescence image of HeLa cells fixed with 4% formaldehyde then stained with Cell Navigator® F-Actin Labeling Kit *Red Fluorescence* in a Costar black 96-well plate. Cells were labeled with iFluor® 594-Phalloidin (Cat#22664, Red) and nuclei stain DAPI (Cat#17507, Blue), respectively. Cell endoplasmic reticulum (ER) was stained with ER Green™ (Cat#22635, Green) before fixation.
Fluorescence image of HeLa cells fixed with 4% formaldehyde then stained with Cell Navigator® F-Actin Labeling Kit *Red Fluorescence* in a Costar black 96-well plate. Cells were labeled with iFluor® 594-Phalloidin (Cat#22664, Red) and nuclei stain DAPI (Cat#17507, Blue), respectively. Cell endoplasmic reticulum (ER) was stained with ER Green™ (Cat#22635, Green) before fixation.
Fluorescence image of HeLa cells fixed with 4% formaldehyde then stained with Cell Navigator® F-Actin Labeling Kit *Red Fluorescence* in a Costar black 96-well plate. Cells were labeled with iFluor® 594-Phalloidin (Cat#22664, Red) and nuclei stain DAPI (Cat#17507, Blue), respectively. Cell endoplasmic reticulum (ER) was stained with ER Green™ (Cat#22635, Green) before fixation.
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Telephone1-800-990-8053
Fax1-800-609-2943
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Spectral properties
Absorbance (nm)587
Correction Factor (260 nm)0.05
Correction Factor (280 nm)0.04
Extinction coefficient (cm -1 M -1)2000001
Excitation (nm)587
Emission (nm)603
Quantum yield0.531
Storage, safety and handling
H-phraseH303, H313, H333
Hazard symbolXN
Intended useResearch Use Only (RUO)
R-phraseR20, R21, R22
UNSPSC12352200
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OverviewpdfSDSpdfProtocol


Absorbance (nm)
587
Correction Factor (260 nm)
0.05
Correction Factor (280 nm)
0.04
Extinction coefficient (cm -1 M -1)
2000001
Excitation (nm)
587
Emission (nm)
603
Quantum yield
0.531
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.

Platform


Fluorescence microscope

ExcitationTexas Red channel
EmissionTexas Red channel
Recommended plateBlack wall/clear bottom

Components


Example protocol


AT A GLANCE

Protocol summary

  1. Prepare samples (microplate wells)
  2. Remove the liquid from the plate
  3. Add 100 µL/well of iFluor™ 594-Phalloidin working solution
  4. Stain the cells at RT for 15 to 60 minutes
  5. Wash the cells
  6. 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

  1. 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.

  2. Rinse the fixed cells 2 – 3 times in PBS.

  3. 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.

  4. Add 100 µL/well (96-well plate) of iFluor™ 594-Phalloidin working solution into the fixed cells.

  5. Stain the cells at room temperature for 15 to 60 minutes.

  6. Rinse cells gently with PBS 2 to 3 times to remove excess dye before plate sealing.

  7. Image cells using a fluorescence microscope with Texas Red channel (Ex/Em = 594/610 nm).

Spectrum


Open in Advanced Spectrum Viewer
spectrum

Spectral properties

Absorbance (nm)587
Correction Factor (260 nm)0.05
Correction Factor (280 nm)0.04
Extinction coefficient (cm -1 M -1)2000001
Excitation (nm)587
Emission (nm)603
Quantum yield0.531

Product Family


NameExcitation (nm)Emission (nm)Extinction coefficient (cm -1 M -1)Quantum yieldCorrection Factor (260 nm)Correction Factor (280 nm)
Cell Navigator® F-Actin Labeling Kit *Blue Fluorescence*3454502000010.9510.830.23
Cell Navigator® F-Actin Labeling Kit *Green Fluorescence*4915167500010.910.210.11
Cell Navigator® F-Actin Labeling Kit *Orange Fluorescence*54155710000010.6710.250.15

Images


Citations


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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
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
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
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
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
Microquantification of cellular and in vitro F-actin by rhodamine phalloidin fluorescence enhancement
Authors: Katanaev VL, Wymann MP.
Journal: Anal Biochem (1998): 185
Interhead distances in myosin attached to F-actin estimated by fluorescence energy transfer spectroscopy
Authors: Ishiwata S, Miki M, Shin I, Funatsu T, Yasuda K, dos Remedios CG.
Journal: Biophys J (1997): 895
A conformational change in F-actin when myosin binds: fluorescence resonance energy transfer detects an increase in the radial coordinate of Cys-374
Authors: Moens PD, dos Remedios CG.
Journal: Biochemistry (1997): 7353
Myosin-induced changes in F-actin: fluorescence probing of subdomain 2 by dansyl ethylenediamine attached to Gln-41
Authors: Kim E, Miller CJ, Motoki M, Seguro K, Muhlrad A, Reisler E.
Journal: Biophys J (1996): 1439
Changes in the distribution of F-actin in the fission yeast Schizosaccharomyces pombe by arresting growth in distilled water: correlative studies with fluorescence and electron microscopy
Authors: Kanbe T, Akashi T, Tanaka K.
Journal: J Electron Microsc (Tokyo) (1994): 20