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Phalloidin alexa 568

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Phalloidin-Alexa-568 is a fluorescent conjugate of the toxin phalloidin, which binds specifically to filamentous actin (F-actin) in cells. It is used as a tool for visualizing and quantifying the distribution of F-actin in various cellular and tissue samples.

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Lab products found in correlation

Sytox green, by Thermo Fisher Scientific (3 mentions) Lsm 510 meta, by Zeiss (2 mentions) Horse serum, by Thermo Fisher Scientific (1 mentions) Mca409s, by Abcam (1 mentions) Spe confocal microscope, by Olympus (1 mentions) 3i spinning disk microscope, by Olympus (1 mentions) Goat serum, by Merck Group (1 mentions) Ab13970, by Abcam (1 mentions) Fluoromount, by Merck Group (1 mentions) Tcs sp5, by Leica (1 mentions) Axio imager a1, by Zeiss (1 mentions) Zen 2, by Zeiss (1 mentions) Vectashield containing dapi, by Vector Laboratories (1 mentions) Axiocam 503, by Zeiss (1 mentions) Fluoview fv1200 confocal laser scanning microscope, by Olympus (1 mentions) Anti human cd54 antibody, by BioLegend (1 mentions) Anti mouse alexa647 antibody, by Thermo Fisher Scientific (1 mentions) Lsrfortessa x 20, by BD (1 mentions) Alexa fluor 488 anti mouse secondary antibody, by Thermo Fisher Scientific (1 mentions) Poly d lysine hydrobromide, by Merck Group (1 mentions)

Close spelling variants of this product

Alexa 488- and Alexa 568-conjugated phalloidin Alexa Fluor 488 or Alexa Fluor 568 phalloidin Alexa Fluor 568 phalloidin Alexa Flour 568 phalloidin FITC- or Alexa 568-conjugated phalloidin Alexa 568-labeled phalloidin Alexa Fluor™ 488/568 Phalloidin Alexa 568-conjugated phalloidin Alexa Fluor 568-coupled phalloidin F-actin (Alexa-Fluor-488– or Alexa-Fluor-568–phalloidin;

8 protocols using phalloidin alexa 568

1

Immunofluorescence Staining of Myelinated Cells

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Cells were fixed with 4% PFA (wt/vol) for 10–15 min and blocked for 30 min in 10% goat serum (Sigma), 2% horse serum (GIBCO), and 0.3% Triton-X-100 at 20–25 °C. Primary antibodies were diluted in blocking solution and applied for 1 h at 20–25 °C, and included mouse anti-MAG (1:100, Millipore; MAB1567), rat anti-MBP (1:250; AbD Serotec; MCA409S), and chicken anti-GFP (1:100; Abcam; ab13970). Cells were incubated with fluorescently conjugated secondary antibodies diluted in blocking solution (1:1000, Life Technologies-Molecular Probes), and in a subset of experiments with Phalloidin-Alexa-568 (1:40; ThermoFisher), for 1 h at 20–25 °C. Slides were counterstained with Hoechst (5 µg ml−1) and coverslipped with Fluoromount-G. Cells were imaged on a Leica SPE confocal microscope (40× objective) or an Olympus 3i Spinning Disk microscope (30× or 60× objectives).
Dillenburg A., Ireland G., Holloway R.K., Davies C.L., Evans F.L., Swire M., Bechler M.E., Soong D., Yuen T.J., Su G.H., Becher J.C., Smith C., Williams A, & Miron V.E. (2018). Activin receptors regulate the oligodendrocyte lineage in health and disease. Acta Neuropathologica, 135(6), 887-906.
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2

Immunofluorescence Staining of Cells

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Parental cells and M13HS hybrid clone cells (2×104) were seeded onto cover slips in the appropriate media for 24 h in a humidified atmosphere at 37 °C and 5% CO2. Cells were fixed with paraformaldehyde (PFA; 4% (w/v) in PBS; 20 min, room temperature), washed twice with PBS and were permeabilized with 1% Triton X-100 ((v/v) in PBS; 5 min, RT). Subsequently, cells were washed again twice with PBS and were stained with Phalloidin-Alexa568 (1 h, room temperature) and SYTOX Green (15 min, room temperature; both dyes from Thermo Fisher Scientific, Bonn, Germany). Samples were washed again two time with PBS, mounted with Fluoromount (Sigma-Aldrich, Taufkirchen, Germany) and were finally analyzed by confocal laser scanning microscopy (Leica TCS SP5; Leica Microsystems, Wetzlar, Germany). Images were processed using ImageJ (imagej.nih.gov/ij/).
Gauck D., Keil S., Niggemann B., Zänker K.S, & Dittmar T. (2017). Hybrid clone cells derived from human breast epithelial cells and human breast cancer cells exhibit properties of cancer stem/initiating cells. BMC Cancer, 17, 515.
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3

Fluorescent Gelatin Coated Microscopy

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BxPC-3 cells were plated on coverslips coated with a layer of 2% type B gelatin (MilliporeSigma) or with Oregon Green-labeled gelatin (ThermoFisher Scientific), essentially as described before (Diaz et al., 2013 (link); Díaz, 2013 (link)); PANC-1 were plated on coverslips coated with type B gelatin and treated with 10% NuSerum supplement (Corning) for 24 h. Cells were fixed with 4% paraformaldehyde for 10 min, blocked in PBS containing 0.1% Triton X-100 and 3% BSA, and incubated overnight at 4 °C in primary antibody diluted 1:500 in PBS containing 0.1% Triton X-100 and 0.3% BSA. Secondary antibodies conjugated with Alexa 488 or Alexa 594, as well as Phalloidin-Alexa 568 (ThermoFisher Scientific) were used at 1:500. Samples were mounted using vectashield containing DAPI (Vector Labs). Images were obtained using a 63× objective in a Zeiss AxioImager A1 equipped with a PRIOR Lumen 200, Axiocam 503 mono camera and ZEN2 software (Zeiss).
Chen Y.C., Baik M., Byers J.T., Chen K.T., French S.W, & Díaz B. (2018). TKS5-positive invadopodia-like structures in human tumor surgical specimens. Experimental and molecular pathology, 106, 17-26.
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4

Fixation and Staining of Cellular Blebs

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Samples, isolated blebs and cells were spun on poly-L-lysine coated 25 mm coverslips by centrifugation at 460 g for 10 min. Cells were fixed with 4% paraformaldehyde (PFA) in PBS for 10 min followed by 10 min permeabilisation with 0.2% Triton X-100 at room temperature. Blebs were fixed with combined permeabilisation-fixation for 6 min with 4% PFA in intracellular buffer with 0.2% Triton X-100 followed by 14 min fixation with 4% PFA in intracellular buffer at room temperature, followed by three washes with PBS. Samples were stained with DAPI and phalloidin–Alexa568 (1:500 dilution, Thermo Fisher Scientific, A12380) for 1 h, followed by three washes with PBS.
Samples were imaged using Olympus FluoView FV1200 Confocal Laser Scanning Microscope using a 60× oil objective (NA 1.4).
Vadnjal N., Nourreddine S., Lavoie G., Serres M., Roux P.P, & Paluch E.K. (2022). Proteomic analysis of the actin cortex in interphase and mitosis. Journal of Cell Science, 135(16), jcs259993.
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5

Integrin and Actin Cytoskeleton Analysis in Lymphoid Cells

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Flow cytometry was performed on peripheral blood mononu-clear cells, LCL, and cultured primary B cells using an LSRFortessa X-20 (BD Biosciences). The results were processed using FlowJo v10 software (TreeStar Inc., St. Ashland, OR, USA). To determine integrin expression at the cell surface, LCL were labeled with an anti-human CD11a antibody (TS2/4; Biolegend) for total CD11a expression, or an anti-human CD11a antibody (Hl111; Biolegend) for inactive/closed conformation-CD11a expression, or an anti-human CD54 antibody (Biolegend), followed by an anti-mouse-Alexa647 antibody (ThermoFisher Scientific). To determine F- and G-actin content in two LCL samples side by side, one sample was incubated with an anti-human CD54 antibody (Biolegend) for 30 min on ice and thereafter labeled with DNaseI-Alexa488 (ThermoFisher Scientific) and phalloidin-Alexa568 (ThermoFisher Scientific).
Record J., Sendel A., Kritikou J.S., Kuznetsov N.V., Brauner H., He M., Nagy N., Oliveira M.M., Griseti E., Haase C.B., Dahlström J., Boddul S., Wermeling F., Thrasher A.J., Liu C., Andersson J., Claesson H.E., Winqvist O., Burns S.O., Björkholm M, & Westerberg L.S. (2020). An intronic deletion in megakaryoblastic leukemia 1 is associated with hyperproliferation of B cells in triplets with Hodgkin lymphoma. Haematologica, 105(5), 1339-1350.
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6

Hypoxia-Reoxygenation Injury and Sevoflurane

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Cells were grown on poly-D-lysine hydrobromide (50–100μg (cm2)-1 Sigma) and collagen-coated glass cover slips (Karl Hecht GmbH&Co KG, Sondheim, Germany) and exposed to normoxia or severe hypoxia prior to 4h reoxygenation with or without sevoflurane as described. The monoclonal mouse anti-β-catenin antibody was used at a dilution of 1:500 (BD), monoclonal mouse anti-ZO-1 antibody at a dilution of 1:100 and phalloidin-Alexa 568 at 1:30 (both Life Technologies). The secondary anti-mouse Alexa-Fluor 488 antibody (Life technologies) was applied at a dilution of 1:500 and 4’, 6-diamidino-2-phenylindole (DAPI, Roche Diagnostics, Mannheim, Germany) for nuclear staining was diluted to 1:1000.
Restin T., Kajdi M.E., Schläpfer M., Roth Z’graggen B., Booy C., Dumrese C, & Beck-Schimmer B. (2017). Sevoflurane protects rat brain endothelial barrier structure and function after hypoxia-reoxygenation injury. PLoS ONE, 12(10), e0184973.
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7

Immunostaining and Confocal Imaging Protocol

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Immunostaining of cell cultures and cryostate sections was performed as described previously (Vutskits et al., 2001 (link)). For phalloidin staining of actin filaments, cells were incubated 20 min RT with phalloidin-Alexa568 (Life Technologies, 1/40) following the incubation with secondary antibodies. Images were acquired with a Nikon Eclipse 80i (Nikon Instruments) epifluorescent microscope, with an LSM 510 META (Carl Zeiss) and a Nikon A1 (Nikon Instruments) laser scanning confocal microscope. Image processing was carried out with the NIS-Elements Advanced Research platform (Nikon Instruments, version 3.21.0), and the Metamorph Software (Molecular Devices, version 7.4).
Egervari K., Potter G., Guzman-Hernandez M.L., Salmon P., Soto-Ribeiro M., Kastberger B., Balla T., Wehrle-Haller B, & Kiss J.Z. (2015). Astrocytes Spatially Restrict VEGF Signaling by Polarized Secretion and Incorporation of VEGF into the Actively Assembling Extracellular Matrix. Glia, 64(3), 440-456.
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8

Immunostaining of HEp-2 Cells

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HEp-2 cells (ATCC) were split 1/3 in T175 on day −2 and seeded to glass coverslips at day −1 (100K/well, 24 well). Cells were fixed with 1%PFA for 20 min at RT. Cells were then blocked and permeabilized with block/perm buffer (2% FBS, 0.1% Tx-100 in PBS). After 30 min, cells were stained o/n in block/perm buffer with indicated antibodies at 10 ug/ml, 1 ug/ml or 0.1 ug/ml (C11 and 564 only). The next day, coverslips were washed 3 times and incubated for one hour with Goat anti Mouse IgG (H+L) Alexa488 (Life Technologies), phalloidin Alexa568 (Life Technologies) and DAPI. Coverslips were then washed 4 times and mounted on slides with Fluorogel as mounting medium. Detectors were set to no primary Ab control. Anti HA Ab 6649 was used as negative control. C11, the original hybridoma from which the 564 BCR was created was used as positive control in addition to re-cloned 564.
Degn S.E., van der Poel C.E., Firl D.J., Ayoglu B., Al Qureshah F.A., Bajic G., Mesin L., Reynaud C.A., Weill J.C., Utz P.J., Victora G.D, & Carroll M.C. (2017). Clonal Evolution of Autoreactive Germinal Centers. Cell, 170(5), 913-926.e19.
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