Phalloidin-iFluor® 594 Conjugate

Fluorescence image of HeLa cells fixed with 4% formaldehyde then stained with Cell Navigator® F-Actin Labeling Kit *Red Fluorescence* in a Costar black 96-well plate. Cells were labeled with Phalloidin-iFluor® 594  (Cat#23122, Red) and nuclei stain DAPI (Cat#17507, Blue), respectively. Cell endoplasmic reticulum (ER) was stained with ER Green™ (Cat#22635, Green) before fixation.

Distribution of MCs in the membranous cochlea. The MCs were scored in each cochlear preparation, and the results are presented as a total number of MCs (non-degranulated and degranulated) per area ((a) n = 7; (b) n = 9). (a) Violin plot demonstrating MCs distribution in the explant areas containing either spiral limbus with OC or the lateral wall; (b) Violin plot demonstrating MCs distribution in the apical, medial, and basal parts of the cochlea; the marker shows the mean. (c–f) Representative micrographs showing the cochlear MCs in the medial part of the cochlea. The explants were stained with phalloidin-iFluor 594, avidin–Alexa Fluor ™ 488, and DAPI. The arrows point to MCs. (f) contains a digital enlargement of the MC image form (e). The data were derived from two independent experiments, and the differences were calculated for means. “ns” indicates not significant (p > 0.05); * p < 0.05; *** p < 0.001. ((a) Kruskal–Wallis test; (b) Mann–Whitney test). Source: <b>Degranulation of Murine Resident Cochlear Mast Cells: A Possible Factor Contributing to Cisplatin-Induced Ototoxicity and Neurotoxicity</b> by Karayay <em>et. al.</em>, <em>Int. J. Mol. Sci.</em> Feb. 2023

Cromolyn used at high concentrations decreases the numbers of IHCs and OHCs. (a) The intact hair cells were scored along the length of 100 µm of the cochlear explant (spiral limbus containing OC), and the percentages of intact IHCs (red circles) and OHCs (green squares) were determined and plotted on the y-axis. The control explants (n = 4) were cultured for 24 h in a tissue culture medium. The treatment groups (n = 4 for each treatment) were cultured for 24 h with cromolyn at the following concentrations: 5 µM, 10 µM, 25 µM, 50 µM, 100 µM, and 200 µM. (b,c) Representative micrograph showing intact (b) and damaged (c) hair cells. Arrows point out intact HCs (white) and damaged HCs (green). Scale bar represents 10 µM. (d–i) Representative micrograph showing the HCs after 24 h of exposure to 5 µM, 10 µM, 25 µM, 50 µM, 100 µM, and 200 µM cromolyn. The cochlea explants were stained with phalloidin-iFluor 594. The scale bar represents 10 µm. Four independent experiments were performed; the data are reported as mean ± SEM. * p < 0.05; ** p < 0.01 (two-way ANOVA with Dunnett multiple comparison test). Source: <b>Degranulation of Murine Resident Cochlear Mast Cells: A Possible Factor Contributing to Cisplatin-Induced Ototoxicity and Neurotoxicity</b> by Karayay <em>et.al.</em>, <em>Int. J. Mol. Sci.</em> Feb. 2023

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Physical properties

Molecular weight	~1600
Solvent	DMSO

Spectral properties

Absorbance (nm)	587
Correction Factor (260 nm)	0.05
Correction Factor (280 nm)	0.04
Extinction coefficient (cm ^-1 M ^-1)	180000¹
Excitation (nm)	588
Emission (nm)	604
Quantum yield	0.53¹

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

~1600

Absorbance (nm)

587

Correction Factor (260 nm)

0.05

Correction Factor (280 nm)

0.04

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

180000¹

Excitation (nm)

588

Emission (nm)

604

Quantum yield

0.53¹

This red fluorescent phalloidin conjugate (equivalent to Alexa Fluor® 594-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™ 594 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 or Texas Red 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™ 594 Conjugate working solution

Add 1 µL of Phalloidin-iFluor™ 594 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™ 594 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 or Texas Red filter set.

Spectrum

Open in Advanced Spectrum Viewer

Spectral properties

Absorbance (nm)	587
Correction Factor (260 nm)	0.05
Correction Factor (280 nm)	0.04
Extinction coefficient (cm ^-1 M ^-1)	180000¹
Excitation (nm)	588
Emission (nm)	604
Quantum yield	0.53¹

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® 555 Conjugate	557	570	100000¹	0.64¹	0.23	0.14
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® 594-streptavidin conjugate	588	604	180000¹	0.53¹	0.05	0.04
Show More (4)

Images

Figure 1. Fluorescence image of HeLa cells fixed with 4% formaldehyde then stained with Cell Navigator® F-Actin Labeling Kit *Red Fluorescence* in a Costar black 96-well plate. Cells were labeled with Phalloidin-iFluor® 594 (Cat#23122, Red) and nuclei stain DAPI (Cat#17507, Blue), respectively. Cell endoplasmic reticulum (ER) was stained with ER Green™ (Cat#22635, Green) before fixation.

Figure 2. Distribution of MCs in the membranous cochlea. The MCs were scored in each cochlear preparation, and the results are presented as a total number of MCs (non-degranulated and degranulated) per area ((a) n = 7; (b) n = 9). (a) Violin plot demonstrating MCs distribution in the explant areas containing either spiral limbus with OC or the lateral wall; (b) Violin plot demonstrating MCs distribution in the apical, medial, and basal parts of the cochlea; the marker shows the mean. (c–f) Representative micrographs showing the cochlear MCs in the medial part of the cochlea. The explants were stained with phalloidin-iFluor 594, avidin–Alexa Fluor ™ 488, and DAPI. The arrows point to MCs. (f) contains a digital enlargement of the MC image form (e). The data were derived from two independent experiments, and the differences were calculated for means. “ns” indicates not significant (p > 0.05); * p < 0.05; *** p < 0.001. ((a) Kruskal–Wallis test; (b) Mann–Whitney test). Source: Degranulation of Murine Resident Cochlear Mast Cells: A Possible Factor Contributing to Cisplatin-Induced Ototoxicity and Neurotoxicity by Karayay et. al., Int. J. Mol. Sci. Feb. 2023

Figure 3. Cromolyn used at high concentrations decreases the numbers of IHCs and OHCs. (a) The intact hair cells were scored along the length of 100 µm of the cochlear explant (spiral limbus containing OC), and the percentages of intact IHCs (red circles) and OHCs (green squares) were determined and plotted on the y-axis. The control explants (n = 4) were cultured for 24 h in a tissue culture medium. The treatment groups (n = 4 for each treatment) were cultured for 24 h with cromolyn at the following concentrations: 5 µM, 10 µM, 25 µM, 50 µM, 100 µM, and 200 µM. (b,c) Representative micrograph showing intact (b) and damaged (c) hair cells. Arrows point out intact HCs (white) and damaged HCs (green). Scale bar represents 10 µM. (d–i) Representative micrograph showing the HCs after 24 h of exposure to 5 µM, 10 µM, 25 µM, 50 µM, 100 µM, and 200 µM cromolyn. The cochlea explants were stained with phalloidin-iFluor 594. The scale bar represents 10 µm. Four independent experiments were performed; the data are reported as mean ± SEM. * p < 0.05; ** p < 0.01 (two-way ANOVA with Dunnett multiple comparison test). Source: Degranulation of Murine Resident Cochlear Mast Cells: A Possible Factor Contributing to Cisplatin-Induced Ototoxicity and Neurotoxicity by Karayay et.al., Int. J. Mol. Sci. Feb. 2023

Citations

View all 105 citations: Citation Explorer

Phosphorylation-dependent recognition of diverse protein targets by the cryptic GK domain of MAGI MAGUKs
Authors: Zhang, Meng and Cao, Aili and Lin, Lin and Chen, Ying and Shang, Yuan and Wang, Chao and Zhang, Mingjie and Zhu, Jinwei
Journal: Science Advances (2023): eadf3295

The human microglial surveillant phenotype is preserved by de novo neurosteroidogenesis through the control of cholesterol homeostasis: Crucial role of 18 kDa Translocator Protein
Authors: Angeloni, Elisa and Germelli, Lorenzo and Marchetti, Laura and Da Pozzo, Eleonora and Tremolanti, Chiara and Wetzel, Christian H and Baglini, Emma and Taliani, Sabrina and Da Settimo, Federico and Martini, Claudia and others,
Journal: Biochimica et Biophysica Acta (BBA)-Molecular Basis of Disease (2023): 166751

Rare disease research workflow using multilayer networks elucidates the molecular determinants of severity in Congenital Myasthenic Syndromes
Authors: N{\'u}{\v{n}}ez-Carpintero, Iker and O'Connor, Emily and Rigau, Maria and Bosio, Mattia and Azuma, Yoshiteru and Topf, Ana and Thompson, Rachel and 't Hoen, Peter AC and Chamova, Teodora and Tournev, Ivailo and others,
Journal: bioRxiv (2023): 2023--01

Characterization of cellular phenotype and proinflammatory response in Meniere’s Disease
Authors: Flook Pereira, Marisa and others,
Journal: (2023)

Rab3A/Rab27A System Silencing Ameliorates High Glucose-Induced Injury in Podocytes
Authors: Martinez-Arroyo, Olga and Flores-Chova, Ana and Sanchez-Garcia, Belen and Redon, Josep and Cortes, Raquel and Ortega, Ana
Journal: Biology (2023): 690

Knockout of miR-184 in zebrafish leads to ocular abnormalities by elevating p21 levels
Authors: Zhang, Jing and Li, Ping and Sun, Luqian and Jiang, Ning and Guo, Wenya and Wang, Jungai and Gao, Fen and Li, Jing and Li, Hui and Zhang, Jun and others,
Journal: The FASEB Journal (2023): e22927

Estrogen ameliorates sepsis-induced vascular hyporeactivity in thoracic aorta of female rats via permissive effect of GR$\alpha$ expression
Authors: Wang, Shan and Wu, Jue and Yang, Kai and Liu, Chunlei and Li, Xin and Wu, Liben and Qi, Xiaoqin and Zhang, Ruizhi and Ni, Wenfeng and Pei, Jinlian and others,
Journal: Biochemical and Biophysical Research Communications (2023)

Biocompatibility of Subperiosteal Dental Implants: Effects of Differently Treated Titanium Surfaces on the Expression of ECM-Related Genes in Gingival Fibroblasts
Authors: Roy, Marco and Corti, Alessandro and Dominici, Silvia and Pompella, Alfonso and Cerea, Mauro and Chelucci, Elisa and Dorocka-Bobkowska, Barbara and Daniele, Simona
Journal: Journal of Functional Biomaterials (2023): 59

Norepinephrine as the Intrinsic Contributor to Contact Lens--Induced Pseudomonas aeruginosa Keratitis
Authors: Zhang, Bi Ning and Qi, Benxiang and Chu, Wai Kit and Song, Fangying and Li, Suxia and Dong, Qiaoqiao and Shao, Zheng and Zhang, Bin and Du, Xianli and Ma, Xiubin and others,
Journal: Investigative Ophthalmology \& Visual Science (2023): 26--26

Degranulation of Murine Resident Cochlear Mast Cells: A Possible Factor Contributing to Cisplatin-Induced Ototoxicity and Neurotoxicity
Authors: Karayay, Bet{\"u}l and Olze, Heidi and Szczepek, Agnieszka J
Journal: International Journal of Molecular Sciences (2023): 4620

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
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Journal: Proc Natl Acad Sci U S A (2010): 20246

Labeling cytoskeletal F-actin with rhodamine phalloidin or fluorescein phalloidin for imaging
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Journal: Cold Spring Harb Protoc (2010): pdb prot4947

Protective effect of bile acid derivatives in phalloidin-induced rat liver toxicity
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Journal: Toxicol Appl Pharmacol (2009): 21

Effect of Phalloidin on Filaments Polymerized from Heart Muscle Adp-Actin Monomers
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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
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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