Alexa 647 conjugated phalloidin

Manufactured by Thermo Fisher Scientific

Sourced in United Kingdom

Alexa Fluor 647-conjugated phalloidin is a fluorescent dye-conjugated phalloidin used for the high-affinity labeling and visualization of F-actin in fixed cells and tissues.

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Lab products found in correlation

4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (4 mentions) Zen blue software, by Zeiss (2 mentions) Alexa 488 secondary antibody, by Thermo Fisher Scientific (2 mentions) Dragonfly confocal microscope, by Oxford Instruments (2 mentions) Anti β catenin antibody, by Thermo Fisher Scientific (2 mentions) Prolong gold antifade reagent, by Thermo Fisher Scientific (2 mentions) Paraformaldehyde (pfa), by Merck Group (2 mentions) Rabbit ki 67, by Thermo Fisher Scientific (1 mentions) Tcs sp8 x confocal microscope, by Leica camera (1 mentions) Rabbit anti phospho histone h3 ser10, by Merck Group (1 mentions) Prolong, by Thermo Fisher Scientific (1 mentions) Mouse anti β galactosidase, by Promega (1 mentions) Rabbit anti cleaved caspase 3, by Cell Signaling Technology (1 mentions) Sp8 confocal microscope, by Leica camera (1 mentions) Bovine thrombin, by Merck Group (1 mentions) Fibrinogen, by Merck Group (1 mentions) Fluorophore conjugated secondary antibody, by Thermo Fisher Scientific (1 mentions) Sq22536, by Bio-Techne (1 mentions) 15d pgj2, by Enzo Life Sciences (1 mentions) Ciglitazone, by Bio-Techne (1 mentions)

20 protocols using alexa 647 conjugated phalloidin

Immunofluorescence Analysis of HepG2 Spheroids

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For 2D-cultured HepG2 cells
immunofluorescence analysis, spheroids generated on gels were fixed
with 3.7% formalin for 30 min, followed by further washes with PBS.
The cells were permeabilized with 0.2% Triton X-100 in PBS for 1 h,
followed by another 1 h of blocking with 2% BSA in PBS. The primary
antibody incubation was performed in 1% BSA in PBS at room temperature
for 2 h. The following primary antibodies were used: mouse β1-integrin
(Santa Cruz, Cat. #sc-53711, 1:200) and rabbit Ki-67 (Thermo scientific,
#rb1510-P0, 1:500). The secondary antibody incubation was in 1% BSA
in PBS at room temperature for 1 h, followed by three PBS washes.
Alexa 488 (Invitrogen, Cat. #A21206, 1:400)- or 555 (Invitrogen, Cat.
#A21424, 1:400)-conjugated secondary antibodies were used. All immunofluorescence
experiments were performed with negative controls without any primary
antibodies. The samples were then incubated with DAPI (5 μg/mL)
at room temperature for 10 min, followed by washing by PBS three times.
The samples were mounted in PBS. Alexa-647-conjugated phalloidin (Life
Technologies) was used 1:100 in 1% BSA to visualize F-actin microfilaments.
Images were acquired using a Leica TCS SP8X confocal microscope. Acquired
images were processed with Fiji, a newer processing package based
on ImageJ.

Liu J., Zhang Y., van Dongen K., Kennedy C., Schotman M.J., Marín San Román P.P., Storm C., Dankers P.Y, & Sijbesma R.P. (2023). Hepatic Spheroid Formation on Carbohydrate-Functionalized Supramolecular Hydrogels. Biomacromolecules, 24(6), 2447-2458.

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RPE Cytoskeleton Imaging in Atrophic AMD

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Of 35 RPE-Bruch’s membrane flat-mounts previously prepared from 35 Caucasian donors^{32 (link)}, 5 nvAMD tissues were revisited. To enable definition of RPE cytoskeleton, F-actin was labeled with Alexa 647 conjugated phalloidin (Life Technologies, Grand Island, NY, USA). To illustrate RPE cell melanosome content and to illustrate F-actin and lipofuscin/melanolipofuscin distribution, bright field and fluorescence imaging, respectively, was performed using a confocal microscope (BX51, Olympus, Center Valley PA USA) at predefined locations and settings, as described^{35 (link)}, plus additional images in areas affected by atrophy.

Zanzottera E.C., Ach T., Huisingh C., Messinger J.D., Freund K.B, & Curcio C.A. (2016). Visualizing retinal pigment epithelium phenotypes in the transition to atrophy in neovascular age-related macular degeneration. Retina (Philadelphia, Pa.), 36(Suppl 1), S26-S39.

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Immunostaining and Confocal Imaging of Drosophila Midguts

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Samples were fixed, immunostained, and mounted as previously described^{3 (link)}. Primary antibodies: mouse anti-β-galactosidase (1:400, Promega Z3781), mouse anti-Armadillo (1:100, DSHB N27A1), rabbit anti-cleaved caspase 3 (1:200, Cell Signaling, generous gift from D. Bilder^{3 (link)}) rabbit anti-diphospho-ERK (1:400, Cell Signaling 4370P), goat anti-HRP-Cy3 (Cappel, 1:100) which stains stem cells and enteroblasts^{3 (link)}, mouse anti-Coracle (1:50, DSHB C615.16), mouse anti-Discs large C615.16 (1:50, DSHB 4F3), and rabbit anti-phospho-histone H3, Ser 10 (1:1000, EMD Millipore). Secondary antibodies: Alexa Fluor 488-, 555- or 647-conjugated donkey anti-rabbit or anti-mouse IgGs (1:800, LifeTechnologies A31570, A11001, and A21244). Nuclei were stained with DAPI (LifeTechnologies, 1:1000). Actin was stained with SiR-Actin (Spiro-chrome, 1:500) or Alexa 647-conjugated phalloidin (1:100, LifeTechnologies). Samples were mounted in ProLong (LifeTechnologies). Imaging of samples was performed on a Leica SP8 confocal microscope, with serial optical sections taken at 3.5 µm intervals through the entirety of whole-mounted, immunostained midguts. Representative images are shown in all panels.

Liang J., Balachandra S., Ngo S, & O’Brien L.E. (2017). Feedback regulation of steady-state epithelial turnover and organ size. Nature, 548(7669), 588-591.

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Platelet Activation and Signaling Pathways

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Fibrinogen, bovine thrombin, H89, GW9662 and IMBX were purchased from Sigma Aldrich (Poole, UK). 15dPGJ2 was purchased from Enzo Life Sciences and Ciglitazone and SQ22536 from Tocris Bioscience (Bristol, UK). The cAMP ELISA kit was from Enzo Life Sciences (Exeter, UK). Primary anti‐ FAK (focal adhesion kinase) (C20), Syk (N‐19), PLCγ2 (Q20), β3 (C20) and actin (C11) antibodies were purchased from Santa Cruz Biotechnology (Heidelberg, Germany). Phospho‐specific primary antibodies for β3 Y779 and Akt S473 were from Abcam (Cambridge, UK). Anti‐phospho–PKC (protein kinase C) substrate, phospho‐S157 and S239 VASP and phospho‐Ser 19 myosin light chain antibodies were purchased from New England BioLabs (Cell Signalling, Hitchin, UK), and anti‐phospho‐Tyr 4G10 antibody was purchased from Millipore (Watford, UK). Fluorophore conjugated secondary antibodies, Fluo‐4 calcium indicator dye and Alexa‐488 and Alexa‐647 conjugated phalloidin were purchased from Life Technologies (Paisely, UK). All other reagents were from previously described sources 26.

Unsworth A.J., Kriek N., Bye A.P., Naran K., Sage T., Flora G.D, & Gibbins J.M. (2017). PPARγ agonists negatively regulate αIIbβ3 integrin outside‐in signaling and platelet function through up‐regulation of protein kinase A activity. Journal of Thrombosis and Haemostasis, 15(2), 356-369.

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Immunocytochemical Analysis of Cellular Processes

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Immunostaining was performed as described^{17 (link)}. Cells were fixed with 4% paraformaldehyde for 30 min and permeabilised in phosphate-buffered saline containing 0.4% saponin, 1% bovine serum albumin and 2% normal goat serum at room temperature for 15 min. Cells were incubated with primary antibodies at 4 °C overnight and then with fluorescent dye-conjugated secondary antibodies (Life Technologies) at room temperature for 1 h. F-actin was visualised with Alexa647-conjugated phalloidin (Life Technologies)^{17 (link),30}. Endocytosis and cholesterol localisation were examined using FITC-dextran (Life Technologies)^{46 (link)} and filipin (SIGMA)^{47 (link)}, respectively. Fluorescence images were acquired with FV-1000 Confocal Microscope (OLYMPUS).

Matsumoto N., Sekiya M., Tohyama K., Ishiyama-Matsuura E., Sun-Wada G.H., Wada Y., Futai M, & Nakanishi-Matsui M. (2018). Essential Role of the a3 Isoform of V-ATPase in Secretory Lysosome Trafficking via Rab7 Recruitment. Scientific Reports, 8, 6701.

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Antibody Utilization for Immunoprecipitation and Imaging

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Antibodies used for immunoprecipitation and immunolabeling include rabbit anti-BIN1 exon 13 (custom made, generous gift from Sarcotein), mouse anti-BIN1 exon 17 (clone 99D, Sigma), recombinant monoclonal anti-BIN1 exon 13 (generous gift from Sarcotein), chicken anti-GFP (Abcam), mouse anti-Flag (Sigma), rabbit anti-CHMP4B (Abcam), rabbit anti-V5 (Sigma), rabbit anti-actin (Sigma), mouse and rabbit anti-GST (Santa Cruz), mouse anti-CD63 (Thermo Scientifics), fluorescein-conjugated anti-PI(4,5)P2 IgM (Echelon Bioscience), and fluorescein-conjugated anti-PI(3,4,5)P3 IgM (Echelon Bioscience). Alexa 647-conjugated WGA, Alexa 488-conjugated annexin V, Alexa 555-conjugated annexin V, Alexa 647-conjugated annexin V, and Alexa 647-conjugated phalloidin were purchased from Life Technologies. For imaging and flow cytometry, recombinant anti-BIN1 exon 13 was conjugated with Alexa 647 using a monoclonal antibody labeling kit (Life Technologies).

Xu B., Fu Y., Liu Y., Agvanian S., Wirka R.C., Baum R., Zhou K., Shaw R.M, & Hong T. (2017). The ESCRT-III pathway facilitates cardiomyocyte release of cBIN1-containing microparticles. PLoS Biology, 15(8), e2002354.

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High-Throughput Imaging of Reprogrammed Colonies

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For convenience, we reproduce below methodological details originally available in^{13 (link)}. Plates were fixed with 4% paraformaldehyde 8 days after induction of reprogramming and then stained with Alexa647-conjugated Phalloidin (Life Technologies). Imaging was performed on a BD Pathway 435 automated imaging system (BD Biosciences). At every xy-position, that is, for every well, 6 images were captured across a z-stack height of 800 μm. For each well, these 6 images in each channel were collapsed into a single additive image.
All images were processed using algorithms developed in CellProfiler v.9777 (Broad Institute). Collapsed image stacks for each well in the AF647 channels were input. In an initial analysis, images were subjected to a threshold and segmented to obtain total colony numbers per well.

Luisier R., Girgin M., Lutolf M.P, & Ranga A. (2020). Mammary epithelial morphogenesis in 3D combinatorial microenvironments. Scientific Reports, 10, 21635.

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Immunoblotting and Immunofluorescence Microscopy

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For immunoblotting, we used anti-PICK1 (Neuromab; University of California, Davis, Davis, CA), glyceraldehyde-3-phosphate dehydrogenase (GAPDH; Abcam, Cambridge, UK), Rac1 (BD Biosciences, San Jose, CA), tubulin (Sigma-Aldrich, Dorset, UK), and horseradish peroxidase–conjugated secondary antibodies (Sigma-Aldrich). Immunoreactive bands were detected by enhanced chemiluminescence. For immunofluorescence microscopy, we used anti-PICK1 (Abcam), anti-Flag (Sigma-Aldrich), and cy3-conjugated secondary antibody (Jackson ImmunoResearch Labs, West Grove, PA). F-actin was visualized with Alexa 647–conjugated phalloidin (Invitrogen, Paisley, UK).

Cockbill L.M., Murk K., Love S, & Hanley J.G. (2015). Protein interacting with C kinase 1 suppresses invasion and anchorage-independent growth of astrocytic tumor cells. Molecular Biology of the Cell, 26(25), 4552-4561.

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Quantifying Immune Cell Activation by Imaging ASC Specks

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Following treatment, the cells were washed once in PBS, then fixed in 2% PFA at 4°C overnight (for BLaER1 ASC speck analysis) or 4% PFA on ice for 30 min. The cells were then washed twice in PBS containing 20 mM Glycine, then twice in PBS. To stain for intracellular targets, the cells were permeabilized in 0.1% Triton X-100 for 5 min and blocked in intracellular staining solution (PBS +10% goat serum, 1% HI-FBS, and 0.1% Triton X-100) for 30 min RT. Next, we used Alexa-647-conjugated Phalloidin (Invitrogen, A22287) for 30 min RT in an intracellular staining solution to stain actin. The cells were then washed (3× 5 min) and incubated with DAPI (1 μg/ml, 10 min) before being washed and imaged. For ASC speck detection, the fixed cells were incubated with DRAQ5 (eBioscience, 65-0880-96) for 5 min (1:2,000 dilution), then imaged directly. All imaging was performed with an Observer.Z1 epifluorescence microscope, 20× objective (dry, PlanApochromat, NA 0.8; ZEISS), Axiocam 506 mono, and ZEN Blue software (ZEISS). Image analysis of all ASC speck experiments was done using a cell profiler pipeline optimized to detect either ASC specks or nuclei. A minimum of 6 images was analyzed for each condition in each experiment.

Mangan M.S., Gorki F., Krause K., Heinz A., Pankow A., Ebert T., Jahn D., Hiller K., Hornung V., Maurer M., Schmidt F.I., Gerhard R, & Latz E. (2022). Transcriptional licensing is required for Pyrin inflammasome activation in human macrophages and bypassed by mutations causing familial Mediterranean fever. PLOS Biology, 20(11), e3001351.

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3D Culture of Anchored Breast Cells

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MCF10A cells were
cultured as described above. Cells were collected and stained with
CellTracker Green CMFDA, as described above or left unstained. The
green and unstained cells were subsequently labeled with anchor strands
1 and 2, respectively, and assembled as described above. Clusters
containing at least 1 green cell were purified from the unassembled
cell population using a FACSAria II (UCSF Laboratory for Cell Analysis)
and grown for 48 h in 3D on-top cultures in 8-well chamber slides
(Lab-Tek) which were performed as previously described using growth-factor-reduced
lrECM lots with protein concentrations between 9 and 11 mg/mL (Matrigel;
BD Biosciences) (Debnath et al., 2003). After 48 h, the 3D cultures
were fixed with 4% Paraformaldehyde in PBS. The 3D cultures were stained,
as previously described by Debnath et al. (2003). Structures were
stained with rat anti-human α₆-integrin antibodies
(Millipore clone NKI-GoH3MAB1378) for the primary and Alexa-568 conjugated
goat antirat antibodies (Invitrogen) for the secondary. Alexa-647
conjugated phalloidin (Invitrogen) and 1× DAPI in PBS was used
to stain the actin cytoskeleton and nuclei, respectively. Confocal
images were acquired on Zeiss Axio Observer Z1 equipped with a Yokogawa
spinning disk unit and an EM-CCD camera.

Weber R.J., Liang S.I., Selden N.S., Desai T.A, & Gartner Z.J. (2014). Efficient Targeting of Fatty-Acid Modified Oligonucleotides to Live Cell Membranes through Stepwise Assembly. Biomacromolecules, 15(12), 4621-4626.

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