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

Fluorescence images of HeLa cells fixed with 4% formaldehyde then stained with Cell Navigator® F-Actin Labeling Kit *Blue Fluorescence* in a Costar black 96-well plate. Cells were labeled with iFluor® 350-Phalloidin (Cat#22660) without (A) or with (B) pre-treatment of phalloidin for 10 minutes.
Fluorescence images of HeLa cells fixed with 4% formaldehyde then stained with Cell Navigator® F-Actin Labeling Kit *Blue Fluorescence* in a Costar black 96-well plate. Cells were labeled with iFluor® 350-Phalloidin (Cat#22660) without (A) or with (B) pre-treatment of phalloidin for 10 minutes.
Fluorescence images of HeLa cells fixed with 4% formaldehyde then stained with Cell Navigator® F-Actin Labeling Kit *Blue Fluorescence* in a Costar black 96-well plate. Cells were labeled with iFluor® 350-Phalloidin (Cat#22660) without (A) or with (B) pre-treatment of phalloidin for 10 minutes.
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Telephone1-800-990-8053
Fax1-800-609-2943
Emailsales@aatbio.com
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Spectral properties
Correction Factor (260 nm)0.83
Correction Factor (280 nm)0.23
Extinction coefficient (cm -1 M -1)200001
Excitation (nm)345
Emission (nm)450
Quantum yield0.951
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


Correction Factor (260 nm)
0.83
Correction Factor (280 nm)
0.23
Extinction coefficient (cm -1 M -1)
200001
Excitation (nm)
345
Emission (nm)
450
Quantum yield
0.951
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 blue fluorescence. The kit uses a blue fluorescent phalloidin conjugate that is selectively bound to F-actins. This blue 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

Excitation350 nm
Emission450 nm
Recommended plateBlack wall/clear bottom
Instrument specification(s)DAPI channel

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™ 350-Phalloidin working solution
  4. Stain cells at RT for 15 to 60 minutes
  5. Wash cells
  6. Examine the specimen under microscope at Ex/Em = 350/450 nm

Important notes
Warm all the components to room temperature before opening.

PREPARATION OF WORKING SOLUTION

Add 10 µL of iFluor™ 350-Phalloidin (Component A) to 10 mL of Labeling Buffer (Component B). Note: Different cell types might be stained differently. The concentration of iFluor™ 350-Phalloidin working solution should be prepared accordingly. Protect from light.

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 1X iFluor™ 350-Phalloidin working solution into the fixed cells, and stain the cells at room temperature for 15 to 60 minutes.

  5. Rinse cells gently with PBS 2 to 3 times to remove excess dye before plate sealing. Image by using the DAPI channel.

Spectrum


Open in Advanced Spectrum Viewer
spectrum

Spectral properties

Correction Factor (260 nm)0.83
Correction Factor (280 nm)0.23
Extinction coefficient (cm -1 M -1)200001
Excitation (nm)345
Emission (nm)450
Quantum yield0.951

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 *Green Fluorescence*4915167500010.910.210.11
Cell Navigator® F-Actin Labeling Kit *Orange Fluorescence*54155710000010.6710.250.15
Cell Navigator® F-Actin Labeling Kit *Red Fluorescence*58760320000010.5310.050.04

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
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
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
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