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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


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)
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
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Images


Citations


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Metformin augments major cytoplasmic organization except for spindle organization in oocytes cultured under hyperglycemic and hyperlipidemic conditions: An in vitro study
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Journal: Toxicology and Applied Pharmacology (2024): 117039
Promoting Biological Similarity by Collagen Microfibers in 3D Colorectal Cancer-Stromal Tissue: Replicating Mechanical Properties and Cancer Stem Cell Markers
Authors: Sasaki, Naoko and Asano, Yoshiya and Sorayama, Yukiko and Kamimura, Chihiro and Kitano, Shiro and Irie, Shinji and Katayama, Ryohei and Shimoda, Hiroshi and Matsusaki, Michiya
Journal: Acta Biomaterialia (2024)
Elevated cancer-associated hyaluronan correlates with diagnosis and lymph node metastasis of papillary thyroid cancer
Authors: Liu, Qinqing and Ye, Jingwen and Liu, Bohan and Guo, Qian and Wang, Siyi and Liu, Yiwen and He, Yiqing and Du, Yan and Zhang, Guoliang and Shen, Yunyue and others,
Journal: Laboratory Investigation (2024): 102104
Mechanistic investigation of wogonin in delaying the progression of endothelial mesenchymal transition by targeting the TGF-$\beta$1 pathway in pulmonary hypertension
Authors: Wang, Xinyue and Cui, Lidan and Wang, Yichen and Zeng, Zuomei and Wang, Hongjuan and Tian, Leiyu and Guo, Jian and Chen, Yucai
Journal: European Journal of Pharmacology (2024): 176786
pH-Responsive Mesoporous Silica Nanoparticles Loaded with Naringin for Targeted Osteoclast Inhibition and Bone Regeneration
Authors: Gong, Shuwei and Lang, Shuang and Wang, Yan and Li, Xiongfeng and Tian, Aixian and Ma, Jianxiong and Ma, Xinlong
Journal: International Journal of Nanomedicine (2024): 6337--6358
Mussel-Derived and Bioclickable Peptide Mimic for Enhanced Interfacial Osseointegration via Synergistic Immunomodulation and Vascularized Bone Regeneration
Authors: Zhou, Wei and Liu, Yang and Dong, Jiale and Hu, Xianli and Su, Zheng and Zhang, Xianzuo and Zhu, Chen and Xiong, Liming and Huang, Wei and Bai, Jiaxiang
Journal: Advanced Science (2024): 2401833
Invasive metastatic tumor-camouflaged ROS responsive nanosystem for targeting therapeutic brain injury after cardiac arrest
Authors: Xia, Yiyang and Zou, Chenming and Kang, Weichao and Xu, Tianhua and Shao, Rongjiao and Zeng, Ping and Sun, Bixi and Chen, Jie and Qi, Yiming and Wang, Zhaozhong and others,
Journal: Biomaterials (2024): 122678
Fenofibrate reduces cardiac remodeling by mitochondrial dynamics preservation in a renovascular model of cardiac hypertrophy
Authors: Castiglioni, Laura and Gelosa, Paolo and Muluhie, Majeda and Mercuriali, Benedetta and Rzemieniec, Joanna and Gotti, Marco and Fiordaliso, Fabio and Busca, Giuseppe and Sironi, Luigi
Journal: European Journal of Pharmacology (2024): 176767
Soft Polyethylene Glycol Hydrogels Support Human PSC Pluripotency and Morphogenesis
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Journal: ACS Biomaterials Science \& Engineering (2024)

References


View all 127 references: Citation Explorer
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
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
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
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
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
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
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
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
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