Phalloidin-iFluor® 488 Conjugate

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Telephone	1-800-990-8053
Fax	1-800-609-2943
Email	sales@aatbio.com
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Physical properties

Molecular weight	~1400
Solvent	DMSO

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 yield	0.9¹

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

~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

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

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

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

Open in Advanced Spectrum Viewer

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 yield	0.9¹

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	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® 488-streptavidin conjugate	491	516	75000¹	0.9¹	0.21	0.11
Show More (4)

Images

Figure 1. 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).

Figure 2. Figure 2. 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.

Figure 3. 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: Opisthorchis viverrini, Clonorchis sinensis and Opisthorchis felineus liver flukes affect mammalian host microbiome in a species-specific manner by Pakharukova et. al., PLoS Negl Trop Dis. Feb. 2023.

Figure 4. 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: 3D bioprinted functional skeletal muscle models have potential applications for studies of muscle wasting in cancer cachexia by Andrea García-Lizarribar et.al., Biomaterials Advances April 2023.

Figure 5. 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: 3D bioprinted functional skeletal muscle models have potential applications for studies of muscle wasting in cancer cachexia by Andrea García-Lizarribar et.al., Biomaterials Advances April 2023.

Myogenic differentiation in 3D bioprinted models. Bioprinted rings after 15 days of culture in GM with differentiated fibers expressing α-actinin (yellow) and MHC (red). F-actin (green) and nuclei (blue) are also stained. 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.

Figure 6. Myogenic differentiation in 3D bioprinted models. Bioprinted rings after 15 days of culture in GM with differentiated fibers expressing α-actinin (yellow) and MHC (red). F-actin (green) and nuclei (blue) are also stained. Scale bar = 200 μm. Actin was stained with Phalloidin-iFluor 488. Source: 3D bioprinted functional skeletal muscle models have potential applications for studies of muscle wasting in cancer cachexia by Andrea García-Lizarribar et.al., Biomaterials Advances April 2023.

Bioprinted human muscle models. Immunostaining of bioprinted rings cultured for 14 days in GM showing Human Skeletal Muscle Myoblasts (HSMM) fibers expressing MHC (red) and α-actinin (yellow). F-actin (green) and nuclei (blue) were also stained. Scale bar = 100 μm. Data is presented as mean ± SD, Unpaired t-test *p-value <0.05, **p-value <0.005 and ***p-value <0.0005. 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.

Figure 7. Bioprinted human muscle models. Immunostaining of bioprinted rings cultured for 14 days in GM showing Human Skeletal Muscle Myoblasts (HSMM) fibers expressing MHC (red) and α-actinin (yellow). F-actin (green) and nuclei (blue) were also stained. Scale bar = 100 μm. Data is presented as mean ± SD, Unpaired t-test *p-value <0.05, **p-value <0.005 and ***p-value <0.0005. Actin was stained with Phalloidin-iFluor 488. Source: 3D bioprinted functional skeletal muscle models have potential applications for studies of muscle wasting in cancer cachexia by Andrea García-Lizarribar et.al., Biomaterials Advances April 2023.

MSCs-ApoEVs promote fusion and apoptosis ratio of C2C12 myoblasts in vitro. The representative fluorescence images of C2C12 myoblasts after MSCs-ApoEVs treatment, MSCs-ApoEVs were pre-stained by PKH26, scale bar indicates 50 μm. Source: <b>MSCs-derived apoptotic extracellular vesicles promote muscle regeneration by inducing Pannexin 1 channel-dependent creatine release by myoblasts</b> by Qingyuan Ye, Xinyu Qiu, Jinjin Wang, Boya Xu, Yuting Su, Chenxi Zheng, Linyuan Gui, Lu Yu, Huijuan Kuang, Huan Liu, Xiaoning He, Zhiwei Ma, Qintao Wang & Yan Jin. <em>International Journal of Oral Science</em>, January 2023.

Figure 8. MSCs-ApoEVs promote fusion and apoptosis ratio of C2C12 myoblasts in vitro. The representative fluorescence images of C2C12 myoblasts after MSCs-ApoEVs treatment, MSCs-ApoEVs were pre-stained by PKH26, scale bar indicates 50 μm. Source: MSCs-derived apoptotic extracellular vesicles promote muscle regeneration by inducing Pannexin 1 channel-dependent creatine release by myoblasts by Qingyuan Ye, Xinyu Qiu, Jinjin Wang, Boya Xu, Yuting Su, Chenxi Zheng, Linyuan Gui, Lu Yu, Huijuan Kuang, Huan Liu, Xiaoning He, Zhiwei Ma, Qintao Wang & Yan Jin. International Journal of Oral Science, January 2023.

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Organotypic 3D Cell-Architecture Impacts the Expression Pattern of miRNAs-mRNAs Network in Breast Cancer SKBR3 Cells
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References

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