Phalloidin-iFluor® 555 Conjugate

Confocal microscopy images of HEp-2 cells incubated with concentrated culture supernatants of Ssp variants (25 μg/mL) for 5 h. Ssp was visualised with anti-Ssp polyclonal antibody followed by Alexa Fluor Plus 647 conjugated secondary antibody (yellow), actin cytoskeleton was stained with iFluor 555 phalloidin (magenta) and nucleus stained with DAPI (cyan). Ssp variants include wildtype (WT), deletions of Loop 2 (ΔL2), deletion of active site protrusions E2 and E3 (ΔE2, ΔE3, ΔE2/ΔE3), and site-directed mutants of the active site Ser (S314A) and RGD motif (RAE). pBAD denotes vector only control. Images are representative of cells observed from at least three independent experiments. Scale bar represents 50 μm. Source: <b>Crystal structure of a subtilisin-like autotransporter passenger domain reveals insights into its cytotoxic function</b>. by Hor, <em>et. al.</em>., <em>Nature Communications</em>, March 2023.

Axl and integrin αvβ5 expression in AKH tumor cells and glioma cell lines. Cells were cultured on glass slides, fixed with formalin, and then post-treated with Triton-X100. iFluor-555 phalloidin (red) and DAPI (blue) were used to visualize the cells and their nuclei. Bound Axl- and integrin αvβ5-specific monoclonal antibodies were detected by using a secondary anti-mouse antibody-Alexa 488 conjugate (green). Scale = 200 µm. Source: <b>Isolation of Cells from Glioblastoma Multiforme Grade 4 Tumors for Infection with Zika Virus prME and ME Pseudotyped HIV-1</b> by Pöhlking <em>et. al.</em>, <em>Int. J. Mol. Sci.</em> Feb. 2023.

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Telephone	1-800-990-8053
Fax	1-800-609-2943
Email	sales@aatbio.com
Quotation	Request
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Shipping	Standard overnight for United States, inquire for international

Physical properties

Molecular weight	~1300
Solvent	DMSO

Spectral properties

Correction Factor (260 nm)	0.23
Correction Factor (280 nm)	0.14
Extinction coefficient (cm ^-1 M ^-1)	100000¹
Excitation (nm)	557
Emission (nm)	570
Quantum yield	0.64¹

Storage, safety and handling

Certificate of Origin	Download PDF
H-phrase	H301, H311, H331
Hazard symbol	T
Intended use	Research Use Only (RUO)
R-phrase	R23, R24, R25
Storage	Freeze (< -15 °C); Minimize light exposure
UNSPSC	12352200

Overview SDS Protocol

Molecular weight

~1300

Correction Factor (260 nm)

0.23

Correction Factor (280 nm)

0.14

Extinction coefficient (cm ^-1 M ^-1)

100000¹

Excitation (nm)

557

Emission (nm)

570

Quantum yield

0.64¹

This orange fluorescent phalloidin conjugate (equivalent to Alexa Fluor® 555-labeled phalloidin) selectively binds to F-actins. 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

Prepare samples in microplate wells
Remove liquid from samples in the plate
Add Phalloidin-iFluor™ 555 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 TRITC 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™ 555 Conjugate working solution

Add 1 µL of Phalloidin-iFluor™ 555 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™ 555 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 TRITC filter set.

Spectrum

Open in Advanced Spectrum Viewer

Spectral properties

Correction Factor (260 nm)	0.23
Correction Factor (280 nm)	0.14
Extinction coefficient (cm ^-1 M ^-1)	100000¹
Excitation (nm)	557
Emission (nm)	570
Quantum yield	0.64¹

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® 488 Conjugate	491	516	75000¹	0.9¹	0.21	0.11
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® 594 Conjugate	588	604	180000¹	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
Phalloidin-iFluor® 700 Conjugate	690	713	220000¹	0.23¹	0.09	0.04
Phalloidin-iFluor® 750 Conjugate	757	779	275000¹	0.12¹	0.044	0.039
Phalloidin-iFluor® 790 Conjugate	787	812	250000¹	0.13¹	0.1	0.09
iFluor® 555-streptavidin conjugate	557	570	100000¹	0.64¹	0.23	0.14
Show More (4)

Images

Figure 1. Fluorescence image of HeLa cells fixed with 4% formaldehyde then stained with Phalloidin-iFluor® 555 Conjugate (Cat#23119, Red) and Nuclear Blue™ DCS1 (Cat#17548, Blue), respectively.

Figure 2. Confocal microscopy images of HEp-2 cells incubated with concentrated culture supernatants of Ssp variants (25 μg/mL) for 5 h. Ssp was visualised with anti-Ssp polyclonal antibody followed by Alexa Fluor Plus 647 conjugated secondary antibody (yellow), actin cytoskeleton was stained with iFluor 555 phalloidin (magenta) and nucleus stained with DAPI (cyan). Ssp variants include wildtype (WT), deletions of Loop 2 (ΔL2), deletion of active site protrusions E2 and E3 (ΔE2, ΔE3, ΔE2/ΔE3), and site-directed mutants of the active site Ser (S314A) and RGD motif (RAE). pBAD denotes vector only control. Images are representative of cells observed from at least three independent experiments. Scale bar represents 50 μm. Source: Crystal structure of a subtilisin-like autotransporter passenger domain reveals insights into its cytotoxic function. by Hor, et. al.., Nature Communications, March 2023.

Figure 3. Axl and integrin αvβ5 expression in AKH tumor cells and glioma cell lines. Cells were cultured on glass slides, fixed with formalin, and then post-treated with Triton-X100. iFluor-555 phalloidin (red) and DAPI (blue) were used to visualize the cells and their nuclei. Bound Axl- and integrin αvβ5-specific monoclonal antibodies were detected by using a secondary anti-mouse antibody-Alexa 488 conjugate (green). Scale = 200 µm. Source: Isolation of Cells from Glioblastoma Multiforme Grade 4 Tumors for Infection with Zika Virus prME and ME Pseudotyped HIV-1 by Pöhlking et. al., Int. J. Mol. Sci. Feb. 2023.

Citations

View all 86 citations: Citation Explorer

Extracellular Matrix Mimicking Dynamic Interpenetrating Network Hydrogel for Skin Tissue Engineering
Authors: Wang, Weibin and Dai, Jiajia and Huang, Yvfeng and Li, Xiaomeng and Yang, Jianmin and Zheng, Yunquan and Shi, Xianai
Journal: Chemical Engineering Journal (2023): 141362

Hydrogels from TEMPO-Oxidized Nanofibrillated Cellulose Support In Vitro Cultivation of Encapsulated Human Mesenchymal Stem Cells
Authors: Nikolits, Ilias and Radwan, Sara and Liebner, Falk and Dietrich, Wolf and Egger, Dominik and Chariyev-Prinz, Farhad and Kasper, Cornelia
Journal: ACS Applied Bio Materials (2023)

Calotropis gigantea incorporated alginate dialdehyde-gelatin hydrogels for cartilage tissue regeneration in Osteoarthritis
Authors: Aswathy, Jalaja and Resmi, Rajalekshmi and Joseph, Josna and Joseph, Roy and John, Annie and Abraham, Annie
Journal: Journal of Drug Delivery Science and Technology (2023): 104372

Characterization of Immortalized Human Prostate Epithelial Cells
Authors: Hashmi, Rumesa
Journal: (2023)

Astragaloside IV Attenuates High-Glucose-Induced Impairment in Diabetic Nephropathy by Increasing Klotho Expression via the NF-$\kappa$B/NLRP3 Axis
Authors: He, Jiaxin and Cui, Jialin and Shi, Yimin and Wang, Tao and Xin, Junyan and Li, Yimeng and Shan, Xiaomeng and Zhu, Zhiyao and Gao, Yanbin and others,
Journal: Journal of Diabetes Research (2023)

Downregulation of HAS-2 regulates the chondrocyte cytoskeleton and induces cartilage degeneration by activating the RhoA/ROCK signaling pathway
Authors: Yang, Junlong and Wang, Liu and Zhang, Zhongjie and Sun, Qing and Zhang, Yuan
Journal: International Journal of Molecular Medicine (2023): 1--14

Isolation of Cells from Glioblastoma Multiforme Grade 4 Tumors for Infection with Zika Virus prME and ME Pseudotyped HIV-1
Authors: P{\"o}hlking, Celine and Beier, Sebastian and Formanski, Jan Patrick and Friese, Michael and Schreiber, Michael and Schwalbe, Birco
Journal: International Journal of Molecular Sciences (2023): 4467

Crystal structure of a subtilisin-like autotransporter passenger domain reveals insights into its cytotoxic function
Authors: Hor, Lilian and Pilapitiya, Akila and McKenna, James A and Panjikar, Santosh and Anderson, Marilyn A and Desvaux, Micka{\"e}l and Paxman, Jason J and Heras, Bego{\~n}a
Journal: Nature Communications (2023): 1163

BMP4 inhibits corneal neovascularization by interfering with tip cells in angiogenesis
Authors: Nan, Weijin and He, Yuxi and Shen, Sitong and Wu, Meiliang and Wang, Shurong and Zhang, Yan
Journal: (2023)

Effects of Various Cell Surface Engineering Reactions on the Biological Behavior of Mammalian Cells
Authors: Cai, Fengying and Ren, Yafeng and Dai, Jiajia and Yang, Jianmin and Shi, Xianai
Journal: Macromolecular Bioscience (2022): 2200379

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