Phalloidin 568

Manufactured by Thermo Fisher Scientific

Sourced in United Kingdom

Phalloidin 568 is a fluorescently labeled phalloidin compound used to stain and visualize F-actin filaments in cells. It binds tightly to F-actin, allowing for the detection and localization of actin cytoskeletal structures.

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Close spelling variants of this product

Alexa Fluor 568-labelled phalloidin (4 citations) Alexa Fluor 568® phalloidin (A12380) (2 citations) Alexa Fluor 568-labeled phalloidin (7 citations) Phalloidin 568 or 647 (2 citations) Alexa Fluor 568 phalloidin (436 citations) Alexa 488- and Alexa 568-conjugated phalloidin (2 citations) Alexa Flour 568 phalloidin (9 citations) Alexa 568-labeled phalloidin (3 citations) BODIPY (558/568) phalloidin (4 citations) FITC- or Alexa 568-conjugated phalloidin (2 citations)

31 protocols using phalloidin 568

Immunocytochemistry for Myosin Heavy Chain

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Following functional analysis, cells were fixed in a 4% paraformaldehyde (PFA) in phosphate buffered saline (PBS) solution for 15 min. Cells were rinsed three times with PBS and then permeabilized in a PBS solution containing 0.1% Triton X-100 for 20 min. Cells were then incubated in a blocking buffer (5% donkey serum with 0.1% bovine serum albumin in PBS) for 20 min. Cells were incubated overnight at 4°C in a primary antibody solution containing the blocking buffer and an antibody against MyHC all classes (DSHB A4.1025) at a 1:10 dilution. On the following day, the primary antibody solution was removed and the cells were rinsed with 3 × 10-min washes in PBS. Cells were then incubated with a secondary antibody solution for 2 hr. This solution contained blocking buffer, donkey-anti-mouse-488 (Invitrogen) diluted 1:250, and phalloidin 568 at 1:40 dilution (Invitrogen). The phalloidin was used for actin filament visualization. After this incubation, cells were rinsed three times with PBS, with 10 min in between each rinse. The second rinse contained a 4′, 6-diamidino-2-phenylindole (DAPI) solution (1:10,000 in PBS; Life Technologies, New York, NY) for visualization of nuclei.

Najjar S.A., Smith A.S., Long C.J., McAleer C.W., Cai Y., Srinivasan B., Martin C., Vandenburgh H.H, & Hickman J.J. (2019). A multiplexed in vitro assay system for evaluating human skeletal muscle functionality in response to drug treatment. Biotechnology and bioengineering, 117(3), 736-747.

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Immunofluorescence Analysis of Chondrocyte Cytoskeleton

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Chondrocytes cultured on glass slides were washed once with PBS−/− and fixed in 4% paraformaldehyde (in PBS−/−) at room temperature for 10 min. Cells were permeabilized with 10% goat serum diluted with 0.2% Triton/PBS−/− for 30 min. Actin staining was performed by incubating samples with DNase I (1:500; Molecular Probes; D12371) to visualize globular actin and Phalloidin-568 (1:20; Invitrogen; A12380) to visualize filamentous actin, in 3% goat serum diluted with 0.2% Triton/PBS−/− for 1 h at room temperature. Vinculin (VCL) staining was performed by incubating samples overnight with the primary antibody (1: 300; Sigma; hVIN-1) diluted in 3% goat serum diluted with 0.2% Triton/PBS−/−. Samples were washed three times and subsequently incubated with the secondary antibody anti-rabbit IgG conjugated Alexa Fluor 488 (1:250; Invitrogen; A11008) and Alexa Fluor 568 phalloidin conjugate (1:20; Invitrogen; A12380) to visualize filamentous actin, in 3% goat serum diluted with 0.2% Triton/PBS−/− for 1 h at room temperature. Samples were counterstained with 4′,6-diamidino-2-phenylindole (1:1500 in PBS−/−) for 5 min. Cells were washed and mounted using Permafluor Mountant (Thermo Scientific; Runcon, Cheshire, UK). Confocal microscopy was performed using a NikonC1si laser scanning confocal microscope.

Delve E., Co V, & Kandel R.A. (2020). Superficial and deep zone articular chondrocytes exhibit differences in actin polymerization status and actin-associated molecules in vitro. Osteoarthritis and Cartilage Open, 2(3), 100071.

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Cytotoxic T Cell Degranulation Assay

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CTLs were washed and allowed to adhere for 12 minutes onto hydrophobic mutliwell slides (Hendles-Essex) in FCS-free IMDM. For conjugation assays, blue P815 target cells were coated with anti-CD3 (UCHT1), plated on multiwall slides in serum free media and CTLs were added for 10-15min. Cells were fixed with 4% paraformaldehyde (15710-S, Electron Microscopy Systems, USA) and incubated in quenching solution (50 mM ammonium chloride in 1X PBS) (15 min at RT) and rinsed twice in PBS. Permeabilization was done with 0.2% Saponin in PBS (5 min, RT) followed by washing and blocking for 30min in blocking buffer (1xPB, 1%BSA, 0.2% saponin). Cells were stained with anti-LAMP1 (H4A3, hybridoma supernatant), anti-RAB27A (abcam, UK) for 1 hour, followed by fluorophore-conjugated secondary antibodies (donkey anti-mouse 488 and goat anti-rabbit 647, Invitrogen, UK) (1 hour at RT) together with phalloidin 568 (Invitrogen, UK). Nuclei were stained for 5 minutes at RT with Hoechst (Thermo Fisher, UK), and samples were mounted in ProLong Diamond Antifade Reagent (Thermo Fisher, UK).

Brauer N., Maruta Y., Lisci M., Strege K., Oschlies I., Nakamura H., Böhm S., Lehmberg K., Brandhoff L., Ehl S., Parvaneh N., Klapper W., Fukuda M., Griffiths G.M., Hennies H.C., Niehues T, & Ammann S. (2023). Immunodeficiency with susceptibility to lymphoma with complex genotype affecting energy metabolism (FBP1, ACAD9) and vesicle trafficking (RAB27A). Frontiers in Immunology, 14, 1151166.

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Fluorescent Visualization of F-Actin

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After blocking with 3% bovine serum albumin (BSA), cells on the coverslip were stained with phalloidin 568 (Invitrogen) to detect F-actin at room temperature for 1 h. Stained coverslips were mounted with 90% glycerol containing DAPI (Invitrogen), and photographed under the immunofluorescent microscope (Olympus).

Hsu T.I., Chen Y.J., Hung C.Y., Wang Y.C., Lin S.J., Su W.C., Lai M.D., Kim S.Y., Wang Q., Qian K., Goto M., Zhao Y., Kashiwada Y., Lee K.H., Chang W.C, & Hung J.J. (2015). A novel derivative of betulinic acid, SYK023, suppresses lung cancer growth and malignancy. Oncotarget, 6(15), 13671-13687.

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Olfactory Neuron Ablation Protocol

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The scheme for ablating olfactory neurons at the placode stage and independent of OR versus IR expression was based on[22 (link)], and the Peb-Gal4, eyFLP recombinant was a gift of Liqun Luo. All animals analyzed were female due to genetic constraints. Animals with eyFLP, Peb-Gal4, and UAS>w+>RTA largely lacked eyes and had visibly affected antennae. shi[ts] was included in the genotype due to exigencies of the crossing strategy. We did not rear flies at the elevated temperature where shi[ts] has an effect on synaptic vesicle release (usually 29°C or higher), and thus do not expect it to have affected the results, which were stark and consistent in additional experiments with ChAT staining where the shi[ts] allele was not included. Brains were dissected at 2–7 days post-eclosion and stained and mounted as described previously[31 (link)]. We analyzed 5–10 brains per genotype per batch, and derived two data-points from each brain corresponding to the two hemispheres. Antibodies were mouse anti-ChAT 9E10 (DSHB, 1:200), mouse nc82 anti-brp (DSHB, 1:40). Brains were counterstained with Phalloidin-568 (Invitrogen, 1:40 or 1:80) and DAPI (1µ g/mL). Images were collected on a Leica SP8 confocal microscope with 1 µm spacing in Z (along the anterior-posterior axis) and ~200 nm axial resolution.

Ryba A., McKenzie S.K., Olivos-Cisneros L., Clowney E.J., Pires P.M, & Kronauer D.J. (2020). Comparative development of the ant chemosensory system. Current biology : CB, 30(16), 3223-3230.e4.

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Fluorescent Protein Internalization Assay

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Cells on coverslips at 70% confluency were incubated with AlexaFluor488-conjugated constructs at 10 μg ml⁻¹ for 1 h at 4 °C. Cells were extensively washed with PBS to remove unbound antibodies and incubated at 37 °C in growth media. Internalization was allowed for 1 h, followed by extensive washing and fixation with 4% formaldehyde for 10 min at 4 °C. Coverslips were then blocked with 5% goat serum in 0.3% Triton-X100 (Sigma). Actin was detected with phalloidin-568 (Invitrogen) and Hoechst trihydrochloride (Invitrogen) was used to stain cell nuclei. Coverslips were mounted on slides using ProLong Gold Antifade (Invitrogen) and examined using Perkin Elmer Spinning Disc Confocal microscope and Volocity Visualization software.

Maruani A., Smith M.E., Miranda E., Chester K.A., Chudasama V, & Caddick S. (2015). A plug-and-play approach to antibody-based therapeutics via a chemoselective dual click strategy. Nature Communications, 6, 6645.

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Immunofluorescence Staining of Drosophila

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Mouse anti-SCAR (DSHB P1C1-SCAR RRID:AB_2618386) was used at a dilution of 1:100. Rat anti HA (Roche catalog number: 11867423001 RRID:AB_390918) was used at a dilution of 1:1000. Guinea pig anti-Non-stop (Mohan et al., 2014a (link)) was used at a dilution of 1:150. Mouse anti-Chaoptin (deposited to the DSHB by Benzer, S and Colley, N.) (DSHB 24B10 RRID:AB_528161) was used at a dilution of 1:250. Goat anti-Rat IGG −568 (Invitrogen Catalog number: A-11077 RRID:AB_141874) was used at a dilution of 1:1000. Goat anti-Mouse IGG-488 (Invitrogen Catalog number: A-11001 RRID:AB_2534069) was used at a dilution of 1:1000. Goat anti-Guinea pig IGG- 488 (Invitrogen Catalog number: A11073 RRID:AB_142018) was used at a dilution of 1:1000. Phalloidin-488 (Invitrogen Catalog number: A12379 RRID:AB_2759222) was used at a dilution of 1:20. Phalloidin-568 (Invitrogen Catalog number: A12380 RRID:AB_2810839) was used at a dilution of 1:20.

Cloud V., Thapa A., Morales-Sosa P., Miller T.M., Miller S.A., Holsapple D., Gerhart P.M., Momtahan E., Jack J.L., Leiva E., Rapp S.R., Shelton L.G., Pierce R.A., Martin-Brown S., Florens L., Washburn M.P, & Mohan R.D. (2019). Ataxin-7 and Non-stop coordinate SCAR protein levels, subcellular localization, and actin cytoskeleton organization. eLife, 8, e49677.

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Protein Fractionation and Detection Protocols

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Western blotting was performed as described previously (Son et al., 2010b (link)). Equal amounts of protein (30 μg/sample) were separated by NuPAGE Bis-Tris electrophoresis system (Invitrogen, Carlsbad, CA) and blotted onto nitrocellulose membrane (Whatman, Dassel, Germany). Blots were probed with primary and then secondary antibodies before exposure to Hyperfilm (Amersham Pharmacia Biotech).
For immunofluorescence analyses, cells were grown on glass cover-slips and treated with Cr(VI) as indicated. Cells were fixed in 4% paraformaldehyde, permeabilized using 0.1% TrtonX-100 and incubated with primary antibody at room temperature. Secondary antibody Alexa Fluor 488 (Invitrogen) and Phalloidin 568 (Invitrogen), and nuclei were counterstained (Vector laboratories, H-1200). Images were acquired using Olympus, BX53.
To generate fractions enriched in nuclear and cytoplasmic proteins, 5 × 10⁶ cells were harvested and washed inPBS. Next, the cells were resuspended in the lysis buffer (10 mM NaCl, 1.5 mM MgCl₂, 10 mM Tris-HCl at 7.5 pH, 0.1 mM PMSF, 1 mM DTT) and were pass through 25G needle 10 times using 1 ml syringe. After this, lysates were left on ice for 20 mins followed by centrifugation at 3000 rpm for 5 min. (nuclear). The nuclear pellets were re-suspended in lysis buffer and then sonicated. The remaining leftover supernatant was considered as cytoplasmic fraction.

Roy R.V., Pratheeshkumar P., Son Y.O., Wang L., Hitron J.A., Divya S.P., Zhang Z, & Shi X. (2016). Different roles of ROS and Nrf2 in Cr(VI)-induced inflammatory responses in normal and Cr(VI)-transformed cells. Toxicology and applied pharmacology, 307, 81-90.

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Immunofluorescence Imaging of T Cell Activation

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Multiwell microscopy slides were cleaned with 70% ethanol for 15 min at room temperature and were coated with 0.01% poly-L-lysine (Millipore Sigma, UK) for 15 min, washed with PBS, and coated with 10 μg/ml hamster humanized anti-CD3 antibody (ChAgly, a gift from Herman Waldmann). Cultured T cells were washed and added in FCS-free IMDM and were allowed to adhere for 12 min. Cells were fixed for 15 min in 4% paraformaldehyde (PFA) (15710-S, Electron Microscopy Systems, USA), permeabilized in 0.1% Triton X-100, and blocked in 2% BSA in PBS (40 min). Samples were labeled for 1 h with primary antibodies [pericentrin (Abcam, UK)] and LAMP1 (H4A3, hybridoma supernatant) and phalloidin followed by fluorophore-conjugated secondary antibodies (donkey anti-mouse 488 and goat anti-rabbit 647, Invitrogen, UK) (1 h at RT) together with phalloidin 568 (Invitrogen, UK). Nuclei were stained for 5 min at RT with Hoechst 33342 (H3570, Thermo Fisher, UK), and samples were mounted in ProLong Diamond Antifade Reagent (P36961, Thermo Fisher, UK). Images were taken with an IX81 Olympus microscope equipped with an Andor Revolution system fitted with a CSU-X1 spinning-disk unit (Yokogawa, UK).

Ohishi Y., Ammann S., Ziaee V., Strege K., Groß M., Amos C.V., Shahrooei M., Ashournia P., Razaghian A., Griffiths G.M., Ehl S., Fukuda M, & Parvaneh N. (2020). Griscelli Syndrome Type 2 Sine Albinism: Unraveling Differential RAB27A Effector Engagement. Frontiers in Immunology, 11, 612977.

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Immunofluorescence Staining of KIAA0100

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Cells were seeded on 8-well cell culture slides and incubated for designated time points and fixed with 4% formaldehyde for 10 min. The slides were then blocked by PBS buffer contains 5% of BSA. Anti-KIAA0100 C-terminal rabbit antibody (ThermoFisher) was co-labeled with anti α-tubulin monoclonal antibody (ThermoFisher) or anti-HSPA1A monoclonal antibody (Abcam) respectively, following by anti-rabbit-Alexa488 and anti-mouse-Alexa555 secondary antibodies, or Phalloidin-568 (ThermoFisher) for F-actin detection respectively. Slides were mounted by Prolong Gold anti-fade mountant with DAPI (4′,6-Diamidino-2-Phenylindole, Dihydrochloride, ThermoFisher). Images were acquired by a Zeiss Axio Imager 2 Fluorescence Microscope as well as Leica TCS SP5 AOBS Spectral Confocal System.

Zhong Z., Pannu V., Rosenow M., Stark A, & Spetzler D. (2018). KIAA0100 Modulates Cancer Cell Aggression Behavior of MDA-MB-231 through Microtubule and Heat Shock Proteins. Cancers, 10(6), 180.

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