Phalloidin-iFluor® 488 Conjugate
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.
![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).](/_next/image?url=https%3A%2F%2Fimages.aatbio.com%2Fproducts%2Ffigures-and-data%2Fphalloidin-ifluor-488-conjugate%2Ffigure-for-phalloidin-ifluor-488-conjugate_Refma.jpg&w=640&q=75)
![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).](/_next/image?url=https%3A%2F%2Fimages.aatbio.com%2Fproducts%2Ffigures-and-data%2Fphalloidin-ifluor-488-conjugate%2Ffigure-for-phalloidin-ifluor-488-conjugate_Refma.jpg&w=640&q=75)
![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).](/_next/image?url=https%3A%2F%2Fimages.aatbio.com%2Fproducts%2Ffigures-and-data%2Fphalloidin-ifluor-488-conjugate%2Ffigure-for-phalloidin-ifluor-488-conjugate_Refma.jpg&w=128&q=25)
![<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’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. Displayed is the Max Intensity Projection of 19 images with 0.2 um spacing in Z.](/_next/image?url=https%3A%2F%2Fimages.aatbio.com%2Fproducts%2Ffigures-and-data%2Fphalloidin-ifluor-488-conjugate%2Ffigure-for-phalloidin-ifluor-488-conjugate_BHKBv.jpg&w=128&q=25)
![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.](/_next/image?url=https%3A%2F%2Fimages.aatbio.com%2Fproducts%2Ffigures-and-data%2Fphalloidin-ifluor-488-conjugate%2Ffigure-for-phalloidin-ifluor-488-conjugate_gc51y.png&w=128&q=25)
![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.](/_next/image?url=https%3A%2F%2Fimages.aatbio.com%2Fproducts%2Ffigures-and-data%2Fphalloidin-ifluor-488-conjugate%2Ffigure-for-phalloidin-ifluor-488-conjugate_X404X.png&w=128&q=25)
![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.](/_next/image?url=https%3A%2F%2Fimages.aatbio.com%2Fproducts%2Ffigures-and-data%2Fphalloidin-ifluor-488-conjugate%2Ffigure-for-phalloidin-ifluor-488-conjugate_nOtF8.png&w=64&q=25)
Example protocol
AT A GLANCE
Protocol Summary
- Prepare samples in microplate wells
- Remove liquid from samples in the plate
- Add Phalloidin-iFluor™ 488 Conjugate solution (100 μL/well)
- Stain the cells at room temperature for 20 to 90 minutes
- Wash the cells
- Examine the specimen under microscope with FITC filter
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
- 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. - Rinse the fixed cells 2–3 times in PBS.
- 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.
- 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.
- 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
Product family
Name | Excitation (nm) | Emission (nm) | Extinction coefficient (cm -1 M -1) | Quantum yield | Correction Factor (260 nm) | Correction Factor (280 nm) |
Phalloidin-iFluor® 350 Conjugate | 345 | 450 | 200001 | 0.951 | 0.83 | 0.23 |
Phalloidin-iFluor® 405 Conjugate | 403 | 427 | 370001 | 0.911 | 0.48 | 0.77 |
Phalloidin-iFluor® 514 Conjugate | 511 | 527 | 750001 | 0.831 | 0.265 | 0.116 |
Phalloidin-iFluor® 532 Conjugate | 537 | 560 | 900001 | 0.681 | 0.26 | 0.16 |
Phalloidin-iFluor® 555 Conjugate | 557 | 570 | 1000001 | 0.641 | 0.23 | 0.14 |
Phalloidin-iFluor® 594 Conjugate | 587 | 603 | 2000001 | 0.531 | 0.05 | 0.04 |
Phalloidin-iFluor® 633 Conjugate | 640 | 654 | 2500001 | 0.291 | 0.062 | 0.044 |
Phalloidin-iFluor® 647 Conjugate | 656 | 670 | 2500001 | 0.251 | 0.03 | 0.03 |
Phalloidin-iFluor® 680 Conjugate | 684 | 701 | 2200001 | 0.231 | 0.097 | 0.094 |
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Citations
View all 337 citations: Citation Explorer
Myeloid cells coordinately induce glioma cell-intrinsic and cell-extrinsic pathways for chemoresistance via GP130 signaling
Authors: Cheng, Jiying and Li, Min and Motta, Edyta and Barci, Deivi and Song, Wangyang and Zhou, Ding and Li, Gen and Zhu, Sihan and Yang, Anru and Vaillant, Brian D and others,
Journal: Cell Reports Medicine (2024)
Authors: Cheng, Jiying and Li, Min and Motta, Edyta and Barci, Deivi and Song, Wangyang and Zhou, Ding and Li, Gen and Zhu, Sihan and Yang, Anru and Vaillant, Brian D and others,
Journal: Cell Reports Medicine (2024)
PAI-1 uncouples integrin-$\beta$1 from restrain by membrane-bound $\beta$-catenin to promote collagen fibril remodeling in obesity-related neoplasms
Authors: Lin, Li-Ling and Nayak, Bijaya and Osmulski, Pawel A and Wang, Exing and Wang, Chen-Pin and Valente, Philip T and Wang, Chiou-Miin and Tan, Xi and Santanam, Nalini and Wang, Tian-Li and others,
Journal: Cell Reports (2024): 114527
Authors: Lin, Li-Ling and Nayak, Bijaya and Osmulski, Pawel A and Wang, Exing and Wang, Chen-Pin and Valente, Philip T and Wang, Chiou-Miin and Tan, Xi and Santanam, Nalini and Wang, Tian-Li and others,
Journal: Cell Reports (2024): 114527
Molecular characterization of breast cancer cell lines in correlation with clinical markers
Authors: Kashyap, D and Salman, H
Journal: Open J Clin Med Images (2024): 1186
Authors: Kashyap, D and Salman, H
Journal: Open J Clin Med Images (2024): 1186
Understanding endogenous oncogenic KRAS mutations in isogenic near-diploid human cell lines derived from hTERT RPE-1
Authors: L Hindul, Naushin
Journal: (2024)
Authors: L Hindul, Naushin
Journal: (2024)
Metformin augments major cytoplasmic organization except for spindle organization in oocytes cultured under hyperglycemic and hyperlipidemic conditions: An in vitro study
Authors: Kunnath, Amrutha Nedumbrakkad and Parker, Shravani Kanakadas and Crasta, Daphne Norma and Kunhiraman, Jyolsna Ponnaratta and Madhvacharya, Vanishree Vasave and Kumari, Sandhya and Nayak, Guruprasad and Lakshmi, R Vani and Modi, Prashanth Kumar and Prasad, TS Keshava and others,
Journal: Toxicology and Applied Pharmacology (2024): 117039
Authors: Kunnath, Amrutha Nedumbrakkad and Parker, Shravani Kanakadas and Crasta, Daphne Norma and Kunhiraman, Jyolsna Ponnaratta and Madhvacharya, Vanishree Vasave and Kumari, Sandhya and Nayak, Guruprasad and Lakshmi, R Vani and Modi, Prashanth Kumar and Prasad, TS Keshava and others,
Journal: Toxicology and Applied Pharmacology (2024): 117039
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
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
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Labeling cytoskeletal F-actin with rhodamine phalloidin or fluorescein phalloidin for imaging
Authors: Chazotte B., undefined
Journal: Cold Spring Harb Protoc (2010): pdb prot4947
Authors: Chazotte B., undefined
Journal: Cold Spring Harb Protoc (2010): pdb prot4947
pH-(low)-insertion-peptide (pHLIP) translocation of membrane impermeable phalloidin toxin inhibits cancer cell proliferation
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Journal: Proc Natl Acad Sci U S A (2010): 20246
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Authors: Luo H, Hallen-Adams HE, Walton JD.
Journal: J Biol Chem (2009): 18070