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AAT Bioquest

Phalloidin-iFluor® 488 Conjugate

Fluorescence images of HeLa cells stained with Phalloidin-iFluor® 488 Conjugate using fluorescence microscope with a FITC filter set (Green). The cells were fixed in 4% formaldehyde, co-labeled with mitochondria dye MitoLite™ Red FX600 (Cat#2677, Red) and Nuclear Blue™ DCS1 (Cat#17548, Blue).
Fluorescence images of HeLa cells stained with Phalloidin-iFluor® 488 Conjugate using fluorescence microscope with a FITC filter set (Green). The cells were fixed in 4% formaldehyde, co-labeled with mitochondria dye MitoLite™ Red FX600 (Cat#2677, Red) and Nuclear Blue™ DCS1 (Cat#17548, Blue).
Fluorescence images of HeLa cells stained with Phalloidin-iFluor® 488 Conjugate using fluorescence microscope with a FITC filter set (Green). The cells were fixed in 4% formaldehyde, co-labeled with mitochondria dye MitoLite™ Red FX600 (Cat#2677, Red) and Nuclear Blue™ DCS1 (Cat#17548, Blue).
<strong>Figure 2.</strong> MDA-MB-231 breast cancer cell grew for 24 h. Cells were stained with Phalloidin-iFluor 488 Conjugate (ATT Bioquest) following manufacturer&rsquo;s instruction. Images were acquired with a 63x/1.4NA objective on a Zeiss laser-scanning confocal microscope by the Advanced Bio-Imaging Facility (ABIF) at McGill.&nbsp;Displayed is the Max Intensity Projection of 19 images with 0.2 um spacing in Z.
Enterobacteriaceae (Lipid A) in the liver of the uninfected and the liver fluke-infected hamsters. a. An uninfected hamster. b. 3D reconstruction of the internal surface of a bile duct after confocal microscopy reveals the presence of Enterobacteriaceae Lipid A. c. Bacteria inside the gut of the O. viverrini parasite. d. Enterobacteriaceae presence inside small bile ducts of an O. viverrini–infected hamster. e. Penetration of bacteria through the injured epithelium in the bile duct of an O. felineus–infected hamster. f. A multilayered epithelium in the bile duct of a C. sinensis–infected hamster. E: epithelial cells; BD: bile duct; red color: Lipid A of Enterobacteriaceae; green color: actin filaments (Phalloidin 488 staining); blue color: nuclei (DAPI staining). E: epithelium of bile duct; BD: bile duct; G: gut of a worm. Source: <b><em>Opisthorchis viverrini</em>, <em>Clonorchis sinensis</em> and <em>Opisthorchis felineus</em> liver flukes affect mammalian host microbiome in a species-specific manner</b> by Pakharukova <em>et. al.</em>, <em>PLoS Negl Trop Dis</em>. Feb. 2023.
Conditioning of GelMA-AlgMA bioinks for skeletal muscle tissue engineering. Modulation of GelMA-AlgMA bioink mechanical properties of GelMA with 0 %,1 % and 2 % AlgMA (n = 9). Confocal images of C2C12 cells after 14 days of differentiation with stained MHC (red), F-actin (green) and nuclei (blue). Actin was stained with Phalloidin-iFluor 488. Scale bar = 100 μm. Source: <b>3D bioprinted functional skeletal muscle models have potential applications for studies of muscle wasting in cancer cachexia</b> by Andrea García-Lizarribar et.al., <em>Biomaterials Advances</em> April 2023.
Myogenic differentiation in 3D bioprinted models. Immunostaining of C2C12 cells after 15 days in bioprinted rings cultured in differentiation medium (DM) and growth medium (GM). Nuclei are stained in blue and green corresponds to F-actin (n = 4). Scale bar = 200 μm. Actin was stained with Phalloidin-iFluor 488. 
Source: <b>3D bioprinted functional skeletal muscle models have potential applications for studies of muscle wasting in cancer cachexia</b> by Andrea García-Lizarribar et.al., <em>Biomaterials Advances</em> April 2023.
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Physical properties
Molecular weight~1400
SolventDMSO
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
Certificate of OriginDownload PDF
H-phraseH301, H311, H331
Hazard symbolT
Intended useResearch Use Only (RUO)
R-phraseR23, R24, R25
StorageFreeze (< -15 °C); Minimize light exposure
UNSPSC12352200

OverviewpdfSDSpdfProtocol


Molecular weight
~1400
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
This green fluorescent phalloidin conjugate (equivalent to Alexa Fluor® 488-labeled phalloidin) selectively binds to F-actins with much higher photostability than the fluorescein-phalloidin conjugates. Used at nanomolar concentrations, phalloidin derivatives are convenient probes for labeling, identifying and quantitating F-actins in formaldehyde-fixed and permeabilized tissue sections, cell cultures or cell-free experiments. Phalloidin binds to actin filaments much more tightly than to actin monomers, leading to a decrease in the rate constant for the dissociation of actin subunits from filament ends, essentially stabilizing actin filaments through the prevention of filament depolymerization. Moreover, phalloidin is found to inhibit the ATP hydrolysis activity of F-actin. Phalloidin functions differently at various concentrations in cells. When introduced into the cytoplasm at low concentrations, phalloidin recruits the less polymerized forms of cytoplasmic actin as well as filamin into stable "islands" of aggregated actin polymers, yet it does not interfere with stress fibers, i.e. thick bundles of microfilaments. The property of phalloidin is a useful tool for investigating the distribution of F-actin in cells by labeling phalloidin with fluorescent analogs and using them to stain actin filaments for light microscopy. Fluorescent derivatives of phalloidin have turned out to be enormously useful in localizing actin filaments in living or fixed cells as well as for visualizing individual actin filaments in vitro. Fluorescent phalloidin derivatives have been used as an important tool in the study of actin networks at high resolution. AAT Bioquest offers a variety of fluorescent phalloidin derivatives with different colors for multicolor imaging applications.

Example protocol


AT A GLANCE

Protocol Summary
  1. Prepare samples in microplate wells
  2. Remove liquid from samples in the plate
  3. Add Phalloidin-iFluor™ 488 Conjugate solution (100 μL/well)
  4. Stain the cells at room temperature for 20 to 90 minutes
  5. Wash the cells
  6. Examine the specimen under microscope with FITC filter 
Important      Warm the vial to room temperature and centrifuge briefly before opening.

Storage and Handling Conditions
The solution should be stable for at least 6 months if store at -20 °C. Protect the fluorescent conjugates from light, and avoid freeze/thaw cycles.
Note     Phalloidin is toxic, although the amount of toxin present in a vial could be lethal only to a mosquito (LD50 of phalloidin = 2 mg/kg), it should be handled with care.

PREPARATION OF WORKING SOLUTION

Phalloidin-iFluor™ 488 Conjugate working solution
Add 1 µL of Phalloidin-iFluor™ 488 Conjugate solution to 1 mL of PBS with 1% BSA.
Note     The stock solution of phalloidin conjugate should be aliquoted and stored at -20 °C. protected from light.
Note     Different cell types might be stained differently. The concentration of phalloidin conjugate working solution should be prepared accordingly.

SAMPLE EXPERIMENTAL PROTOCOL

Stain the cells
  1. Perform formaldehyde fixation. Incubate 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 Phalloidin-iFluor™ 488 Conjugate working solution into the fixed cells, and stain the cells at room temperature for 20 to 90 minutes.
  5. Rinse cells gently with PBS 2 to 3 times to remove excess phalloidin conjugate before plating, sealing and imaging under microscope with FITC filter set. 

Spectrum


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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)
Phalloidin-iFluor® 350 Conjugate3454502000010.9510.830.23
Phalloidin-iFluor® 405 Conjugate4034273700010.9110.480.77
Phalloidin-iFluor® 514 Conjugate5115277500010.8310.2650.116
Phalloidin-iFluor® 532 Conjugate5375609000010.6810.260.16
Phalloidin-iFluor® 555 Conjugate55757010000010.6410.230.14
Phalloidin-iFluor® 594 Conjugate58760320000010.5310.050.04
Phalloidin-iFluor® 633 Conjugate64065425000010.2910.0620.044
Phalloidin-iFluor® 647 Conjugate65667025000010.2510.030.03
Phalloidin-iFluor® 680 Conjugate68470122000010.2310.0970.094
Phalloidin-iFluor® 700 Conjugate69071322000010.2310.090.04
Phalloidin-iFluor® 750 Conjugate75777927500010.1210.0440.039
Phalloidin-iFluor® 790 Conjugate78781225000010.1310.10.09
iFluor® 488-streptavidin conjugate4915167500010.910.210.11
Show More (4)

Images


Citations


View all 306 citations: Citation Explorer
KUS121, a VCP modulator, has an ameliorating effect on acute and chronic heart failure without calcium loading via maintenance of intracellular ATP levels
Authors: Tsuji, Shuhei and Otani, Chiharu and Horie, Takahiro and Watanabe, Shin and Baba, Osamu and Sowa, Naoya and Ide, Yuya and Kashiwa, Asami and Makiyama, Takeru and Imai, Hirohiko and others,
Journal: Biomedicine \& Pharmacotherapy (2024): 115850
Downregulation of the CD151 protects the cardiac function by the crosstalk between the endothelial cells and cardiomyocytes via exosomes
Authors: Jiang, Luying and Liu, Jingbo and Yang, Zhenjia and Wang, Jianyu and Ke, Wenkai and Zhang, Kaiyue and Zhang, Chunran and Zuo, Houjuan
Journal: Plos one (2024): e0297121
Elucidating the distinctive regulatory effects and mechanisms of active compounds in Salvia miltiorrhiza Bunge via network pharmacology: Unveiling their roles in the modulation of platelet activation and thrombus formation
Authors: Zhang, Ying and Xin, Guang and Zhou, Qilong and Yu, Xiuxian and Feng, Lijuan and Wen, Ao and Zhang, Kun and Wen, Tingyu and Zhou, Xiaoli and Wu, Qiuling and others,
Journal: Toxicology and Applied Pharmacology (2024): 116871
Mechanical stretch leads to increased caveolin-1 content and mineralization potential in extracellular vesicles from vascular smooth muscle cells
Authors: Shaver, Mohammad and Gomez, Kassandra and Kaiser, Katherine and Hutcheson, Joshua D
Journal: BMC Molecular and Cell Biology (2024): 1--12
Designed modular protein hydrogels for biofabrication
Authors: Dranseike, Dalia and Ota, Yusuke and Edwardson, Thomas GW and Guzzi, Elia A and Hori, Mao and Nakic, Zrinka Raguz and Deshmukh, Dhananjay V and Levasseur, Mikail D and Mattli, Kevin and Tringides, Christina M and others,
Journal: Acta Biomaterialia (2024)
Endothelial leakiness elicited by amyloid protein aggregation
Authors: Li, Yuhuan and Ni, Nengyi and Lee, Myeongsang and Wei, Wei and Andrikopoulos, Nicholas and Kakinen, Aleksandr and Davis, Thomas P and Song, Yang and Ding, Feng and Leong, David Tai and others,
Journal: Nature Communications (2024): 613
Micro-alloying of Zn and Ca in Vacuum Induction Casted Bioresorbable Mg System: Perspectives on Corrosion Resistance, Cytocompatibility, and Inflammatory Response
Authors: Behera, Manisha and Denys, Agnes and Shabadi, Rajashekhara and Allain, Fabrice and Gruescu, Cosmin
Journal: (2024)
Quantitative Investigation of Intestinal Drug Absorption Enhancement by Drug-Rich Nanodroplets Generated via Liquid--Liquid Phase Separation
Authors: Yoshikawa, Etsushi and Ueda, Keisuke and Hakata, Rei and Higashi, Kenjirou and Moribe, Kunikazu
Journal: Molecular Pharmaceutics (2024)
3D bioprinting of mouse pre-osteoblasts and human MSCs using bioinks consisting of gelatin and decellularized bone particles
Authors: Kara {\"O}zenler, Aylin and Distler, Thomas and Akkineni, Ashwini Rahul and Tihminlioglu, Funda and Gelinsky, Michael and Boccaccini, Aldo R
Journal: Biofabrication (2024)
Harnessing Mechanical Stress with Viscoelastic Biomaterials for Periodontal Ligament Regeneration
Authors: Zhang, Jiu-Jiu and Li, Xuan and Tian, Yi and Zou, Jie-Kang and Gan, Dian and Deng, Dao-Kun and Jiao, Chen and Yin, Yuan and Tian, Bei-Min and Wu, Rui-Xin and others,
Journal: Advanced Science (2024): 2309562

References


View all 127 references: Citation Explorer
Improved penile histology by phalloidin stain: circular and longitudinal cavernous smooth muscles, dual-endothelium arteries, and erectile dysfunction-associated changes
Authors: Lin G, Qiu X, F and el TM, Albersen M, Wang Z, Lue TF, Lin CS.
Journal: Urology (2011): 970 e1
Phalloidin perturbs the interaction of human non-muscle myosin isoforms 2A and 2C1 with F-actin
Authors: Diensthuber RP, Muller M, Heissler SM, Taft MH, Chizhov I, Manstein DJ.
Journal: FEBS Lett (2011): 767
pH-(low)-insertion-peptide (pHLIP) translocation of membrane impermeable phalloidin toxin inhibits cancer cell proliferation
Authors: An M, Wijesinghe D, Andreev OA, Reshetnyak YK, Engelman DM.
Journal: Proc Natl Acad Sci U S A (2010): 20246
Labeling cytoskeletal F-actin with rhodamine phalloidin or fluorescein phalloidin for imaging
Authors: Chazotte B., undefined
Journal: Cold Spring Harb Protoc (2010): pdb prot4947
Protective effect of bile acid derivatives in phalloidin-induced rat liver toxicity
Authors: Herraez E, Macias RI, Vazquez-Tato J, Hierro C, Monte MJ, Marin JJ.
Journal: Toxicol Appl Pharmacol (2009): 21
Effect of Phalloidin on Filaments Polymerized from Heart Muscle Adp-Actin Monomers
Authors: Vig A, Dudas R, Kupi T, Orban J, Hild G, Lorinczy D, Nyitrai M.
Journal: J Therm Anal Calorim (2009): 721
In vitro inhibition of OATP-mediated uptake of phalloidin using bile acid derivatives
Authors: Herraez E, Macias RI, Vazquez-Tato J, Vicens M, Monte MJ, Marin JJ.
Journal: Toxicol Appl Pharmacol (2009): 13
Processing of the phalloidin proprotein by prolyl oligopeptidase from the mushroom Conocybe albipes
Authors: Luo H, Hallen-Adams HE, Walton JD.
Journal: J Biol Chem (2009): 18070
Pygmy squids and giant brains: mapping the complex cephalopod CNS by phalloidin staining of vibratome sections and whole-mount preparations
Authors: Wollesen T, Loesel R, Wanninger A.
Journal: J Neurosci Methods (2009): 63
Anti-acetylated tubulin antibody staining and phalloidin staining in the starlet sea anemone Nematostella vectensis
Authors: Genikhovich G, Technau U.
Journal: Cold Spring Harb Protoc (2009): pdb prot5283