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

Fluorescence image of HeLa cells fixed with 4% formaldehyde then stained with Cell Navigator® F-Actin Labeling Kit *Green Fluorescence* in a Costar black 96-well plate. Cell were labeled with iFluor® 488-Phalloidin (Cat#22261, Green) and nuclei stain DAPI (Cat#17507, Blue), respectively. Cell endoplasmic reticulum (ER) was stained with ER Red™ (Cat#22636, Red) before fixation.
Fluorescence image of HeLa cells fixed with 4% formaldehyde then stained with Cell Navigator® F-Actin Labeling Kit *Green Fluorescence* in a Costar black 96-well plate. Cell were labeled with iFluor® 488-Phalloidin (Cat#22261, Green) and nuclei stain DAPI (Cat#17507, Blue), respectively. Cell endoplasmic reticulum (ER) was stained with ER Red™ (Cat#22636, Red) before fixation.
Ordering information
Price ()
Catalog Number22661
Unit Size
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Additional ordering information
Telephone1-408-733-1055
Fax1-408-733-1304
Emailsales@aatbio.com
InternationalSee distributors
ShippingStandard overnight for United States, inquire for international
Spectral properties
Correction Factor (260 nm)0.21
Correction Factor (280 nm)0.11
Extinction coefficient (cm -1 M -1)750001
Excitation (nm)491
Emission (nm)516
Quantum yield0.91
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)
0.21
Correction Factor (280 nm)
0.11
Extinction coefficient (cm -1 M -1)
750001
Excitation (nm)
491
Emission (nm)
516
Quantum yield
0.91
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 green fluorescence. The kit uses a green fluorescent phalloidin conjugate that is selectively bound to F-actins. This green fluorescent phalloidin conjugate is a high-affinity probe for F-actins with much higher photostability than the fluorescein-phalloidin conjugates. 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

ExcitationFITC filter
EmissionFITC filter
Recommended plateBlack wall/clear bottom

Components


Component A: iFluor™ 488-Phalloidin1 vial (50 µL)
Component B: Labeling Buffer1 bottle (50 mL)

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™ 488-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 = 490/520 nm (FITC filter set)

Important notes
Thaw all the components at room temperature before starting the experiment.

PREPARATION OF WORKING SOLUTION

Add 10 μL of iFluor™ 488-Phalloidin (Component A) to 10 mL of Labeling Buffer (Component B) to make 1X iFluor™ 488-Phalloidin working solution. Protect from light. Note: Different cell types might be stained differently. The concentration of iFluor™ 488-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™ 488-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 FITC filter set (Ex/Em = 490/520 nm).

Spectrum


Open in Advanced Spectrum Viewer
spectrum

Spectral properties

Correction Factor (260 nm)0.21
Correction Factor (280 nm)0.11
Extinction coefficient (cm -1 M -1)750001
Excitation (nm)491
Emission (nm)516
Quantum yield0.91

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 *Orange Fluorescence*54155710000010.6710.250.15
Cell Navigator® F-Actin Labeling Kit *Red Fluorescence*58860418000010.5310.050.04

Citations


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Authors: Shan, Zhongshu and Zhao, Yanyan and Qiu, Zhixue and Angxiu, Suonan and Gu, Yong and Luo, Junming and Bi, Hongtao and Luo, Wei and Xiong, Rui and Ma, Siqing and others,
Journal: Annals of Translational Medicine (2021)
MicroRNA-activated hydrogel scaffold generated by 3D printing accelerates bone regeneration
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Journal: Bioactive Materials (2021)
CHPF Regulates the Aggressive Phenotypes of Hepatocellular Carcinoma Cells via the Modulation of the Decorin and TGF-$\beta$ Pathways
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Journal: Cancers (2021): 1261
Phosphorylated Chitosan Hydrogels Inducing Osteogenic Differentiation of Osteoblasts via JNK and p38 Signaling Pathways
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Authors: Lin, Chun-Shiang and Lin, Chia-Liang and Ying, Tsung-Ho and Chiou, Hui-Ling and Hung, Chia-Hung and Liao, Wei-Shan and Hsieh, Yi-Hsien and Kao, Shao-Hsuan
Journal: Journal of Cellular Physiology (2020)
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Authors: Lin, Zefeng and Cao, Yannan and Zou, Jianming and Zhu, Fangyong and Gao, Yufeng and Zheng, Xiaofei and Wang, Huajun and Zhang, Tao and Wu, Tingting
Journal: Materials Science and Engineering: C (2020): 111032
A novel extrusion-microdrilling approach to fabricate calcium phosphate-based bioceramic scaffolds enabling fast bone regeneration
Authors: He, Fupo and Lu, Teliang and Fang, Xibo and Feng, Songheng and Feng, Shenglei and Tian, Ye and Li, Yanhui and Zuo, Fei and Deng, Xin and Ye, Jiandong
Journal: ACS Applied Materials \& Interfaces (2020)
Collagen-based materials combined with microRNA for repairing cornea wounds and inhibiting scar formation
Authors: Zhao, Xuan and Song, Wenjing and Chen, Yawei and Liu, Sa and Ren, Li
Journal: Biomaterials science (2019): 51--62
Modification of honeycomb bioceramic scaffolds for bone regeneration under the condition of excessive bone resorption
Authors: He, Fupo and Lu, Teliang and Fang, Xibo and Tian, Ye and Li, Yanhui and Zuo, Fei and Ye, Ji and ong, undefined
Journal: Journal of Biomedical Materials Research Part A (2019)

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
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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
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