Phalloidin 633

Manufactured by Thermo Fisher Scientific

Sourced in United States, United Kingdom

Phalloidin-633 is a fluorescent dye that selectively binds to F-actin, a component of the cytoskeleton. It can be used to visualize and study the distribution and organization of actin filaments in cells.

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Lab products found in correlation

4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (4 mentions) Lsm 710 confocal microscope, by Zeiss (3 mentions) Ab14748, by Abcam (2 mentions) Vimentin, by Santa Cruz Biotechnology (1 mentions) α sma, by Agilent Technologies (1 mentions) Harmony high content analysis software, by PerkinElmer (1 mentions) Recombinant human tgf β1, by Thermo Fisher Scientific (1 mentions) 4 6 diamidino 2 phenylindole (dapi), by Vector Laboratories (1 mentions) A1 confocal microscope, by Nikon (1 mentions) Fei xl30 feg sem, by Thermo Fisher Scientific (1 mentions) Dulbecco s modified eagle medium, by Merck Group (1 mentions) Mesenpro rs, by Thermo Fisher Scientific (1 mentions) Tcs spii microscope, by Leica (1 mentions) Ix71 microscope, by Olympus (1 mentions) Anti dlg 4f3, by Developmental Studies Hybridoma Bank (1 mentions) Fv1000, by Olympus (1 mentions) Muc5ac, by Novus Biologicals (1 mentions) Normal goat serum (ngs), by Jackson ImmunoResearch (1 mentions) Tx 100, by Thermo Fisher Scientific (1 mentions) Alexa fluor 555 goat anti rabbit, by Thermo Fisher Scientific (1 mentions)

Close spelling variants of this product

Alexa Fluor 633-conjugated phalloidin (19 citations) Alexa Fluor 633-conjugated Phalloidin and DAPI (2 citations) Phalloidin-conjugated to Alexa Fluor 633 (2 citations) Alexa-633-phalloidin (12 citations) Alexa Fluor™ 488 or 633 Phalloidin (2 citations) Alexa-633-coupled phalloidin (2 citations) OrAlexa-Fluor 633 phalloidin (2 citations) Alexa Fluor 633 phalloidin (78 citations) Phalloidin-AlexaFluor 633 (9 citations) Alexa Fluor-conjugated Phalloidin-546 or-633 (2 citations)

13 protocols using phalloidin 633

Cardiac Fibroblast Characterization and Activation

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Cardiac fibroblasts were isolated from myocardial surgical specimens, as previously described [28 (link),60 (link)]. The cells were characterized by immunostaining following overnight incubation at 4 °C with the primary antibodies: α-SMA (1:100) (Dako, Glostrupe, Denmark), Vimentin (1:100) (Santa Cruz Biotechnology), DDR2 (1:100) (Abcam, Cambridge, UK), and type I collagen antibody (1:100) (Santa Cruz Biotechnology). Next, secondary antibodies and probes were used, with 1 h incubation at room temperature: Phalloidin 633 (ThermoFisher Scientific), anti-mouse IgG Alexa fluor 488-conjugated or anti-goat IgG Alexa fluor 488-conjugated, both diluted at 1:800 (ThermoFisher Scientific). The staining was performed with 4,6-diamidino-2-phenylindole (DAPI) (Vector Laboratories, Burlingame, CA, USA). The A1+ confocal microscope (Nikon, Tokyo, Japan) was used for analysis.
Cardiac fibroblasts were incubated with 10 ng/mL recombinant human TGF-β1 (Peprotech, Rocky Hill, NJ, USA) for 24 h at 37 °C incubation and 5% CO₂ for fibroblast activation. Type I collagen (COL1A1) gene expression was evaluated by RT-qPCR, as described above. Protein expression was analyzed with the Operetta High Content Screening, using the Harmony high-content analysis software (Perkin Elmer, Waltham, MA, USA).

Nonaka C.K., Macêdo C.T., Cavalcante B.R., de Alcântara A.C., Silva D.N., Bezerra M.D., Caria A.C., Tavora F.R., Neto J.D., Noya-Rabelo M.M., Rogatto S.R., Ribeiro dos Santos R., Souza B.S, & Soares M.B. (2019). Circulating miRNAs as Potential Biomarkers Associated with Cardiac Remodeling and Fibrosis in Chagas Disease Cardiomyopathy. International Journal of Molecular Sciences, 20(16), 4064.

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Engineered Adipose Tissue Scaffold

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Human adipose-derived stem cells recovered from the lipoaspirate of one patient at the Adipose Stem Cell Research Laboratory (University of Pittsburgh, Pittsburgh, Pa.) were expanded in MesenPRO RS (Thermo Fisher) growth medium (37°C, 5% carbon dioxide) until 85 percent confluence was obtained. Allograft adipose matrix, reconstituted in basal medium and Dulbecco’s Modified Eagle Medium (Sigma-Aldrich) at 25 percent weight/volume, was placed into 24-well transwell culture inserts in a 3-mm layer. Human adipose-derived stem cells were seeded atop the matrix, 0.2 M cells per 7-mm diameter. Confocal microscopy was performed using a Leica TCS SPII microscope (Leica, Allendale, N.J.). Fixed human adipose-derived stem cell–seeded allograft adipose matrix was stained with Phalloidin 633 and 4′,6-diamidino-2-phenylindole (all from Thermo Fisher), to visualize attachment and stretching at day 3. Lipid content was visualized by boron-dipyrromethene (Thermo Fisher), at days 7, 14, and 21 after cell seeding. Scanning electron microscopy was performed using FEI XL30 FEG-SEM (FEI, Hillsboro, Ore.) at 5-kV accelerating voltage on days 0, 7, and 14. Immunohistochemistry against collagen VI, collagen IV, and laminin and the adipogenic markers adiponectin, leptin, and FABP4 was performed at days 0, 7, and 14. Imaging was performed using an Olympus IX71 microscope (Olympus, Center Valley, Pa.).

Kokai L.E., Schilling B.K., Chnari E., Huang Y.C., Imming E.A., Karunamurthy A., Khouri R.K., D’Amico R.A., Coleman S.R., Marra K.G, & Rubin J.P. (2019). Injectable Allograft Adipose Matrix Supports Adipogenic Tissue Remodeling in the Nude Mouse and Human. Plastic and Reconstructive Surgery, 143(2), 299-309.

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Drosophila Metathoracic Leg Immunohistochemistry

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Metathoracic legs were cut at the coxal segment and fixed in 4% formaldehyde for 45 min prior to dissection. After washing 3× in PBS, a cuticle patch along the femur was removed exposing underlying tissue and samples were post-fixed for 15 min in 4% formaldehyde. Antibody staining protocols followed standard methods and samples were mounted in slow-fade diamond mount (Molecular Probes) (Patel, 1994 (link)). Antibodies and stains used were anti-horse-radish peroxidase (#115-035-166, Jackson ImmunoResearch, 1:550), anti-dlg (4F3, Developmental Studies Hybridoma Bank, 1:200), phalloidin-633 (Molecular Probes, 1:1000), mouse anti-FK2 monoclonal (#BML-PW0150-0025, Enzo Life Sciences, 1:200); mouse anti-Bruchpilot monoclonal (nc82, Developmental Studies Hybridoma Bank, 1:20), mouse anti-ATP5a (#ab14748, Abcam, 1:200). Imaging was performed by confocal microscopy (Olympus FV1000) capturing z-stacks for all samples. Image processing occurred in ImageJ (FIJI) and consisted of cropping images for areas of interest and altering brightness/contrast as needed for publication. Where necessary image size was increased using linear transformation. Image stitching was performed in ImageJ as needed (Preibisch et al., 2009 (link)).

Agudelo A., St. Amand V., Grissom L., Lafond D., Achilli T., Sahin A., Reenan R, & Stilwell G. (2020). Age-dependent degeneration of an identified adult leg motor neuron in a Drosophila SOD1 model of ALS. Biology Open, 9(10), bio049692.

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Immunocytochemistry of Newborn Piglet Trachea

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Trachea were excised from newborn piglets and immediately fixed in 4% paraformaldehyde (EMS) in PBS for 1 hr at room temperature. Tissues were then placed in 30% sucrose and incubated overnight at 4°C, followed by quick-freezing in OCT using a dry ice/EtOH bath and stored at −80°C. Prior to immunocytochemistry, frozen blocks of tissue were cryosectioned at 7 μm followed by permeabilization in 0.3% TX-100 (Thermo-Fisher) in PBS for 20 min, and blocked in Super-Block (Thermo-Fisher) with 5% normal goat serum (Jackson ImmunoResearch) for 1 hr, all at room temperature. Tissue sections were then incubated for 2 hr at 37°C with indicated antibodies: β-tubulin IV(1:300, Biogenex), MUC5AC (1:5000, Novus Biologicals), MUC5B (1:2000, Santa Cruz). Sections were then incubated for 1 hr with secondary antibodies goat-anti-mouse Alexa-Fluor-488 and goat anti-rabbit Alexa-Fluor-555 (1:1000, Molecular Probes/Invitrogen) and phalloidin-633 (1:300, Molecular Probes/Invitrogen). Slides were imaged on an Olympus Fluoview FV3000 confocal microscope with a Plan.ApoN 60X oil lens. Images were post-processed using the Olympus imaging software, CellSens.

Ostedgaard L.S., Price M.P., Whitworth K.M., Abou Alaiwa M.H., Fischer A.J., Warrier A., Samuel M., Spate L.D., Allen P.D., Hilkin B.M., Romano Ibarra G.S., Ortiz Bezara M.E., Goodell B.J., Mather S.E., Powers L.S., Stroik M.R., Gansemer N.D., Hippee C.E., Zarei K., Goeken J.A., Businga T.R., Hoffman E.A., Meyerholz D.K., Prather R.S., Stoltz D.A, & Welsh M.J. (2020). Lack of airway submucosal glands impairs respiratory host defenses. eLife, 9, e59653.

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Visualizing CD28 Receptor Dynamics at the Immunological Synapse

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15 × 10³ murine Dap3/B7 or RAW 264.7 cells were adhered on cover glasses (12 mm) overnight at 37 °C. CH7C17 cells expressing hCD28 WT, or mCD28 WT, or CD28P²¹²A mutant, or chimera CD28 WT, or chimera CD28 A²¹⁰P mutant were transfected for 24 h with 20 μg of GFP or cherry constructs, seeded on cover glasses for 15 min at 37 °C, fixed by 2% paraformaldehyde and permeabilised by 0.1% saponin in PBS containing 1% BSA. Filamentous actin (F-Actin) was stained by using phalloidin-633 (#A22284, 1:75 dilution, Molecular Probes). Confocal observations were performed using a Leica DMIRE (Leica Microsystems, Heidelberg, Germany) and a Zeiss LSM 780 (Zeiss, Berlin, Germany). Images were analysed with the Adobe^® Photoshop^® programme. The relative recruitment index (RRI) was calculated as previously described by the formula: RRI = [mean fluorescence intensity (MFI) at synapse−background]/[MFI at all the cell membrane not in contact with APC−background]. At least 15 cells or conjugates were examined quantitatively for each experiment. Statistical significance was calculated using a Student’s t-test. Signals from different fluorescent probes were taken in parallel. Several cells were analysed for each labelling condition, and representative results are presented.

Porciello N., Grazioli P., Campese A.F., Kunkl M., Caristi S., Mastrogiovanni M., Muscolini M., Spadaro F., Favre C., Nunès J.A., Borroto A., Alarcon B., Screpanti I, & Tuosto L. (2018). A non-conserved amino acid variant regulates differential signalling between human and mouse CD28. Nature Communications, 9, 1080.

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Fluorescent Actin Staining for TNTs

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Fluorescent staining of actin was done with phalloidin 633 (4 units/mL, Molecular Probes) in culture medium containing 0.05% Triton X. The staining was performed on live cells with 15 min incubation at 37°C prior to fixation. The number of TNTs in the actin-labelled populations was counted and expressed as the number of TNTs per 100 cells.

Li C.J., Chen P.K., Sun L.Y, & Pang C.Y. (2017). Enhancement of Mitochondrial Transfer by Antioxidants in Human Mesenchymal Stem Cells. Oxidative Medicine and Cellular Longevity, 2017, 8510805.

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3D Collagen IV Degradation Assay

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3D culturing was performed as previously described with slight modifications [27 , 52 (link)]. Matrigel (BD Bioscience) supplemented with 25 μg/mL DQ-Collagen IV (Molecular Probes) was applied to glass coverslips and allowed to solidify at 37°C for 30 minutes. 1×10⁵ cells were seeded on top of the 3D matrix in 2 mL complete media. Cells were allowed to settle for 2 days prior to treatment with either 100 ng/mL EGF or 33 ng/mL HGF for 2 additional days. To terminate the experiment, the culture was fixed for 30 minutes in warm 4% PFA at 37°C. Actin was then stained using 1:200 Phalloidin 633 (Molecular Probes) for 30 minutes in warm BSP at 37°C. Three fields from three independent experiments were imaged and representative images are shown. To quantify cleaved DQ-Collagen IV signal, the area of the actin cytoskeleton was subtracted from the DQ-Collagen IV fluorescent signal using Image Calculator (ImageJ). Remaining extracellular DQ-Collagen IV fluorescence was assessed by integrated density (ImageJ) and displayed as arbitrary units.

Dykes S.S., Gray A.L., Coleman D.T., Saxena M., Stephens C.A., Carroll J.L., Pruitt K, & Cardelli J.A. (2016). The Arf-like GTPase Arl8b is essential for three-dimensional invasive growth of prostate cancer in vitro and xenograft formation and growth in vivo. Oncotarget, 7(21), 31037-31052.

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Immunofluorescence Microscopy of Abscission Proteins

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All live and immunofluorescence microscopy was performed on a Nikon Eclipse Ti inverted fluorescence microscope. Image capturing was obtained using a Hamamatsu camera controller C10600 and Volocity imaging software, version 6.3 (PerkinElmer; Waltham, MA, USA). Infected cells were fixed in 3.7% paraformaldehyde (Ted Pella) for 15 m, then permeabilized with 0.1% Triton-X-100 (Fisher), blocked with 1% BSA-PBS (Fisher), and stained. Antibodies/dyes were obtained from the following sources: mouse anti-ALIX antibody from Santa Cruz biotechnology (Dallas, TX, USA), rabbit anti-VPS4 from Sigma-Aldrich (St. Louis, MO, USA), mouse anti-CEP55 from Santa Cruz Biotechnology, rabbit anti-CHMP4B from Santa Cruz Biotechnology, CellLight ER-RFP from Thermo Fisher (Waltham, MA, USA), Phalloidin 633, donkey anti-goat 488 from Invitrogen (Waltham, MA), DAPI, goat anti-mouse 488 from Thermo Fisher, anti-GFP 488, FM4-64 from Molecular Probes (Eugene, OR, USA), mouse anti-C. trachomatis LPS donated by Bob Suchland (University of Washington, WA, USA). Staining of abscission proteins: HeLa cells were seeded onto glass chamber slides and fixed with paraformaldehyde 24 h after seeding to stain for antibody localization of CHMP4B, VPS4, CEP55 and ALIX proteins. All abscission proteins are stained with antibodies labeled by GFP and nuclei is displayed by DAPI staining.

Zuck M, & Hybiske K. (2019). The Chlamydia trachomatis Extrusion Exit Mechanism Is Regulated by Host Abscission Proteins. Microorganisms, 7(5), 149.

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Antibody-Mediated Regulation of Cell Signaling

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Anti-E-cadherin (HECD-1) antibody was from Invitrogen. Anti-CAR (H300), anti-TNFR1 (H5), anti-ICAM1 and anti-PKCδ antibodies were from Santa Cruz (Germany). Anti-phospho-PKCδ was from Cell Signalling Technology (USA). p-CAR thr290/ser293 polyclonal antibody was previously described in Morton et al.15 (link), and was developed by Perbioscience (Thermofisher) using the peptide Ac- RTS(pT)AR(pS)YIGSNH-C and was affinity purified before use. Anti-TNFR1 blocking antibody (Mab225) was obtained from R&D Systems (USA). Anti-GFP antibody was from Roche (UK). Anti-mouse HRP and anti-rabbit-HRP were from DAKO, anti-mouse-568, anti-rabbit-568 and phalloidin-633 were all obtained from Invitrogen (UK). CalyculinA, sodium orthovanadate and PI-3-kinase inhibitor (LY294002) were obtained from Calbiochem. TNFα, IL-5, IL-1β, IL-13 and IL-17 were obtained from Sigma-Aldrich (UK). FK was produced and purified as previously described35 (link). PKCδ targeted siRNA and non-targeting controls were obtained from Ambion (USA).

Morton P.E., Hicks A., Ortiz-Zapater E., Raghavan S., Pike R., Noble A., Woodfin A., Jenkins G., Rayner E., Santis G, & Parsons M. (2016). TNFα promotes CAR-dependent migration of leukocytes across epithelial monolayers. Scientific Reports, 6, 26321.

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NF-κB Activation in Cytokine-Treated Cells

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Cells were grown in 8-well chamber slides (NUNC – Thermo Scientific) in normal growth medium and subsequently treated in serum-free medium supplemented with TNF-α (10 ng/mL) for 30 min at 37°. Cells were rinsed with PBS and fixed with paraformaldehyde. After rinsing, they were permeabilized with Triton X-100 (0.2% v/v) at room temperature for 10 min, blocked with 3% (w/v) BSA for 40 min at room temperature then incubated with anti-p65 (Santa Cruz Biotechnology – sc109) in a 1:200 dilution at 4°C overnight. Slides then were washed and incubated with a goat anti-rabbit Alexa-Fluor 488 secondary antibody (Invitrogen) in a 1:200 dilution, DAPI (Invitrogen) in a 1:500 dilution, and Phalloidin 633 (Invitrogen) at a 1:500 dilution for 40 min at room temperature. Slides were mounted in SlowFade Gold (Invitrogen) and imaged using a LSM 710 confocal microscope (Zeiss, Oberkochen, Germany).

Warren C.R., Grindel B.J., Francis L., Carson D.D, & Farach-Carson M.C. (2014). Transcriptional Activation by NFκB Increases Perlecan/HSPG2 Expression in the Desmoplastic Prostate Tumor Microenvironment. Journal of cellular biochemistry, 115(7), 1322-1333.

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