Tetramethylrhodamine isothiocyanate tritc phalloidin

Manufactured by Merck Group

Tetramethylrhodamine isothiocyanate (TRITC)-phalloidin is a fluorescent conjugate used for the detection and visualization of filamentous actin (F-actin) in cells. It binds specifically to F-actin, allowing for the identification and localization of this cytoskeletal component.

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Lab products found in correlation

Triton x 100, by Merck Group (2 mentions) Fv10 asw viewer software, by Olympus (1 mentions) Facsverse flow cytometer, by BD (1 mentions) Fv1000, by Olympus (1 mentions) Ab64693, by Abcam (1 mentions) A31572, by Thermo Fisher Scientific (1 mentions) Fluorescein labeled con a, by Vector Laboratories (1 mentions) Obt1524, by Bio-Rad (1 mentions) β actin clone ac 15, by Merck Group (1 mentions) Anti erm 3142, by Cell Signaling Technology (1 mentions) Pp1α c 19, by Santa Cruz Biotechnology (1 mentions) Gfp trap, by Proteintech (1 mentions) Anti mcherry, by Abcam (1 mentions) Anti ha clone 6e2, by Cell Signaling Technology (1 mentions) Anti gfp jl 8, by Takara Bio (1 mentions) Anti γ tubulin gtu 88, by Merck Group (1 mentions) α tubulin clone dm1a, by Merck Group (1 mentions) Flag m2, by Merck Group (1 mentions) Paraformaldehyde (pfa), by Carlo Erba (1 mentions) Charge coupled device camera, by Zeiss (1 mentions)

Close spelling variants of this product

Phalloidin-tetramethylrhodamine B isothiocyanate (phalloidin-TRITC; Tetramethylrhodamine isothiocyanate (TRITC)-conjugated phalloidin Phalloidin-tetramethylrhodamine B isothiocyanate (TRITC) Phalloidin-Tetramethylrhodamine (TRITC) Tetramethylrhodamine isothiocyanate (TRITC) Phalloidin-tetramethylrhodamine isothiocyanate Tetramethylrhodamine (TRITC) Tetramethylrhodamine–phalloidin Phalloidin-TRITC

10 protocols using tetramethylrhodamine isothiocyanate tritc phalloidin

F-actin Staining of Macrophages Infected with B. abortus

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For F-actin staining, pre-treated macrophages were prepared in 12-well plates with 18-mm diameter glass coverslips and were infected with fluorescein isothiocyanate (FITC)-conjugated B. abortus as previously described [20 (link)]. Macrophages were viewed under a laser scanning confocal microscope (Olympus FV1000; Olympus, Japan) and images were processed by using FV10-ASW Viewer software (ver. 3.1; Olympus).
For fluorescence-activated cell sorting (FACS) analysis, cells in six-well plates were harvested after pre-treatment and infection as described above. Lysophosphatidylcholine (20 µg/mL) containing tetramethylrhodamine isothiocyanate (TRITC)-phalloidin (1 µM) was used to permeabilize and stain the infected cells (both purchased from Sigma-Aldrich) followed by incubation for 30 min at 22℃. After final washing, the F-actin content was quantified by using a FACSVerse flow cytometer (BD Biosciences, USA).

Reyes A.W., Hop H.T., Arayan L.T., Huy T.X., Min W., Lee H.J., Chang H.H, & Kim S. (2018). Tannic acid-mediated immune activation attenuates Brucella abortus infection in mice. Journal of Veterinary Science, 19(1), 51-57.

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Multicolor Immunofluorescence Staining

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The following antibodies were used as primary antibodies: human IgM purified immunoglobulins (1:10 dilution; OBT1524; AbD Serotec), rabbit monoclonal IgG against GS (1:100; 15B1; Cell Signaling), IgMs contained as a contaminant in the OX52 antibody (obtained from mouse ascites), rabbit polyclonal IgG against mannose receptor CD206 (1:1,200; ab64693, Abcam), mouse monoclonal IgG₁ against CD35 (1:40; E11; Thermo Scientific), and mouse monoclonal IgG₁ against FcµR (1:150; OTI1E6; Thermo Scientific).
The following antibodies were used as secondary antibodies: Alexa Fluor (AF) 488 goat anti-human IgM heavy chain (1:200; A-21215; Life Technologies), AF488 goat anti-mouse IgM µ chain specific (1:250; 115-545-075; Jackson ImmunoResearch Laboratories), AF555 donkey anti-rabbit IgG directed against both heavy and light chains (1:250; A-31572; Life Technologies), and AF555 goat anti-mouse IgG₁ (1:250; A-21125; Life Technologies).
Fluorescein-labeled Con A (Vector Laboratories) was incubated overnight instead of primary antibodies in some immunohistochemical experiments in order to detect CA.
Tetramethyl rhodamine-isothiocyanate (TRITC)-phalloidin (Sigma-Aldrich) was used to stain F-actin from macrophages. This staining was combined with the immunohistochemical procedures and was performed by adding TRITC-phalloidin (1:500) to the secondary antibodies.

Riba M., Augé E., Campo-Sabariz J., Moral-Anter D., Molina-Porcel L., Ximelis T., Ferrer R., Martín-Venegas R., Pelegrí C, & Vilaplana J. (2019). Corpora amylacea act as containers that remove waste products from the brain. Proceedings of the National Academy of Sciences of the United States of America, 116(51), 26038-26048.

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Antibody-based Western Blotting and Immunofluorescence

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The following antibodies were used for Western blotting (WB) and IF: anti–γ-tubulin GTU88 (IF), α-tubulin clone DM1A (IF+WB), α-tubulin clone YL 1/2 (IF), β-actin clone AC15 (IF), GFP (WB), PPP1R13L HPA041231 (WB), myosin 1c HPA 001768 (IF, WB), FLAG M2 (WB) from Sigma-Aldrich; anti-mCherry (WB) and NuMA [EP3976] (WB) from Abcam; anti-iASPP 18590–1-AP (WB) from Proteintech and PCRP-PPP1R13L-2G4 (WB) from Developmental Studies Hybridoma Bank; anti–ERM #3142 (WB), anti-EB1 clone 5 (WB), and anti-HA clone 6E2 (WB) from Cell Signaling Technology; anti-EB1 KT51 (IF), myosin Ic (13; WB), PP1α (C-19; WB) from Santa Cruz Biotechnology; anti-CDK5RAP2, A300-554 (IF) from Bethyl; anti–Phospho-Histone H3 (Ser10) from Millipore (WB); and anti-GFP JL-8 from Clontech (for GFP1-10, WB). GFP-Trap was from Chromotek, anti-HA agarose conjugated beads, tetramethylrhodamine isothiocyanate (TRITC)-phalloidin from Sigma-Aldrich, and Strep-Tactin beads from IBA.

Mangon A., Salaün D., Bouali M.L., Kuzmić M., Quitard S., Thuault S., Isnardon D., Audebert S., Puech P.H., Verdier-Pinard P, & Badache A. (2021). iASPP contributes to cell cortex rigidity, mitotic cell rounding, and spindle positioning. The Journal of Cell Biology, 220(12), e202012002.

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Immunofluorescence Staining of Fibroblasts

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Control (untreated or mCNF1‐treated) and CNF1‐treated fibroblasts, seeded on glass coverslips, were fixed with 3.7% paraformaldehyde (Carlo Erba), permeabilized with Triton 0.5% X‐100 (Sigma‐Aldrich, St. Louis) and then incubated at 37°C for 30 min with Tetramethylrhodamine‐isothiocyanate (TRITC)‐phalloidin (Sigma‐Aldrich, cat. n. P1951), diluted 1:100 in PBS for actin cytoskeleton staining. Nuclei were stained for 5 min at RT with 0.2 μg/ml Hoechst 33258 (Sigma‐Aldrich, cat. n. 94,403). To stain mitochondria, cells were incubated with 1 μM Mitotracker Red CMXRos (Invitrogen, Waltham, cat. n. M7512), 30 min before fixation with paraformaldehyde. To stain p62, cells were incubated with Anti‐p62/SQSTM1primary antibody (Sigma‐Aldrich, cat. n. P0067) for 30 min and, following extensive washing, incubated with the appropriate secondary antibody (Alexa Fluor Thermofisher Scientific Invitrogen, cat n. A‐11034). Finally, glass slides were observed with an Olympus BX51/BX52 fluorescence optical microscope (Olympus Corporation of the Americas, Center Valley) equipped with a charge‐coupled device camera (Carl Zeiss). Images were acquired using the IAS 2000 (Delta Systems Inc., Streetsboro) programme.

Cittadini C., Germinario E.A., Maroccia Z., Cosentino L., Maselli V., Gambardella L., Giambenedetti M., Guidotti M., Travaglione S., Fallerini C., Renieri A., Marcillo D.I., Ricceri L., Fortini P., De Filippis B., Fiorentini C, & Fabbri A. (2023). Effects of the Rho GTPase‐activating toxin CNF1 on fibroblasts derived from Rett syndrome patients: A pilot study. Journal of Cellular and Molecular Medicine, 27(10), 1315-1326.

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Immunostaining of Cultured Neurons

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Immunostaining of cultures was performed as described (Ma et al., 2008b (link)). Neurons were fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS). After permeabilization with 0.20% Triton X-100 in blocking buffer (1% bovine serum albumin [BSA], 5% normal goat and/or donkey serum in PBS, pH 7.4), followed by incubation for 50 min in blocking buffer at room temperature, cells were doubly or triply stained with appropriate primary antibodies overnight at 4°C. Primary antibodies were visualized with appropriate secondary antibodies: Cy3-donkey anti-mouse, fluorescein isothiocyanate (FITC)–donkey anti-rabbit, FITC-donkey anti-rat, and FITC-donkey anti-guinea pig immunoglobulin G (IgG) were from Jackson ImmunoResearch Laboratories (West Grove, PA); Alexa Fluor 633–goat anti-rabbit and anti-mouse IgG were from Molecular Probes (Eugene, OR). Images were obtained using a Zeiss (Jena, Germany) LSM510 confocal microscope. Hoechst nuclear stain (Invitrogen, Grand Island, NY) and tetramethylrhodamine isothiocyanate (TRITC)–phalloidin (Sigma-Aldrich, St. Louis, MO) were used where indicated.

Ma X.M., Miller M.B., Vishwanatha K.S., Gross M.J., Wang Y., Abbott T., Lam T.T., Mains R.E, & Eipper B.A. (2014). Nonenzymatic domains of Kalirin7 contribute to spine morphogenesis through interactions with phosphoinositides and Abl. Molecular Biology of the Cell, 25(9), 1458-1471.

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Visualizing Actin Cytoskeleton and Bacterial Pedestals

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HeLa cells were fixed with formalin buffer solution (containing 4% w/v formaldehyde, Sigma) for 20 min at RT and then washed twice with D-PBS. The fixed cells were permeabilized with 0.05% Triton X-100 for 5 min. After washing with PBS three times, the cells were blocked with PBS containing 2% BSA for 10 min. The cells were stained with 1 μg/ml tetramethylrhodamine-isothiocyanate (TRITC)-phalloidin (Sigma) for 15 min at room temperature to visualize actin cytoskeleton and pedestals. After three washes with PBS, the cells were stained with 4'-6-diamidino-2 phenylindole (DAPI, 300 nM) for 5 min at room temperature to visualize bacteria. After three washes with PBS, the coverslips were allowed to dry at room temperature during 20 min and mounted using 5 μl of mounting medium containing 100 mM Tris-HCl (pH 8.5), 10% Mowiol 4–88 (Calbiochem), and 25% glycerol with phenylnediamine (Sigma). Micrographs were taken using a Nikon Eclipse TE 200-U fluorescence microscope equipped with a Hamamatsu camera. Images were processed with Adobe Photoshop. Quantification of pedestals was done by counting the number of pedestals of attached bacteria for a total of 40 infected cells in representative microscope fields.

Mohamed-Salem R., Rodríguez Fernández C., Nieto-Pelegrín E., Conde-Valentín B., Rumbero A, & Martinez-Quiles N. (2019). Aqueous extract of Hibiscus sabdariffa inhibits pedestal induction by enteropathogenic E. coli and promotes bacterial filamentation in vitro. PLoS ONE, 14(3), e0213580.

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Antibody Labeling and Protein Isolation

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Anti-myc (4A6) was purchased from Millipore (Billerica, MA, USA), anti-GFP from Roche Diagnostics (Manheim, Germany), anti-Rab11 from Invitrogen (Carlsbad, CA, USA), anti-b-tubulin, (TUB 2.1) and anti-c-tubulin (GTU-88) from Sigma-Aldrich (St. Louis, MO, USA), anti-transferrin receptor from Zymed Laboratories (South San Francisco, CA, USA) and anti-Golgin-97 and Alexa-conjugated secondary antibodies from Invitrogen. The anti-LMTK1A antibodies have been described previously (Tsutsumi et al. 2008 (Tsutsumi et al. , 2010)) . Tetramethylrhodamine isothiocyanate (TRITC) phalloidin was purchased from Sigma-Aldrich. HRP-conjugated anti-rabbit IgG and HRP-conjugated anti-mouse IgG were purchased from DAKO (Glostrup, Denmark). 4-(2-Aminoethyl)-benzenesulfonylfluoride hydrochloride (AEBSF) was purchased from Sigma-Aldrich, leupeptin from Peptide Institute (Osaka, Japan), DAPI from Dojindo (Kumamoto, Japan) and Protein-G Sepharose from GE Healthcare Bio-Science (Uppsala, Sweden).

Sharma G., Tsutsumi K., Saito T., Asada A., Ando K., Tomomura M, & Hisanaga S.I. (2016). Kinase activity of endosomal kinase LMTK1A regulates its cellular localization and interactions with cytoskeletons. Genes to cells : devoted to molecular & cellular mechanisms, 21(10).

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Immunolocalization of Cathepsin L Proteases in Liver Fluke

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Polyclonal antibodies against recombinant ppFhCL3 and recombinant FhCL3 zymogen were obtained from rabbits immunised with each of the respective recombinant proteins (Eurogentec). F. hepatica NEJs 6 h and 24 h post-excystment were fixed in 4% paraformaldehyde (Sigma-Aldrich) for 1 h at room temperature (RT). After three washes with antibody diluent (AbD: 0.1% [v/v] Triton X-100, 0.1% bovine serum albumin and 0.1% [w/v] sodium azide in 1x PBS), the NEJs were probed with either anti-ppFhCL3, anti-FhCL3 or rabbit pre-immune antiserum diluted 1:500 in AbD buffer, overnight (ON) at 4 °C. After three washes in AbD, the secondary antibody, fluorescein isothiocyanate (FITC)-labelled goat anti-rabbit IgG (Sigma-Aldrich) (1:200), was added and incubated ON at 4 °C. Phalloidin-tetramethylrhodamine isothiocyanate (TRITC) (200 μg/mL [w/v]) (Sigma-Aldrich) was used to counter-stain the muscle tissue and provide structure. All the specimens were whole-mounted in a glycerol solution containing 0.1 M propyl gallate and visualised in a confocal scanning laser microscopy (CSLM) (Leica TCS SP5) under the HCX PL APO CS 100x oil objective lens, using a Leica type F immersion oil.

Pritsch I.C., Tikhonova I.G., Jewhurst H.L., Drysdale O., Cwiklinski K., Molento M.B., Dalton J.P, & Verissimo C.D. (2020). Regulation of the Fasciola hepatica newly excysted juvenile cathepsin L3 (FhCL3) by its propeptide: a proposed ‘clamp-like’ mechanism of binding and inhibition. BMC Molecular and Cell Biology, 21, 90.

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Subcellular Actin Localization in Macrophages

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To analyze the subcellular localization of actin, different human macrophage subtypes were cultured in the presence of storage buffer (vehicle control) or 10 μg/ml recombinant human galectin-2 at day 7 on ibitreat chamber slides (Ibidi, Planegg/Martinsried, Germany) at a density of 1 x 10⁶ cells/ml in DMEM complete medium. Macrophages were fixed in 4% PFA in HBSS (Invitrogen) at RT for 10 minutes, and permeabilized with 0.1% Triton X-100 (Merck) in PBS at RT for 5 minutes. Finally, the cells were stained with 0.4 μg/ml phalloidin-tetramethyl rhodamine iso-thiocyanate (TRITC; Sigma-Aldrich) at RT for one hour. Imaging was performed by a confocal laser scanning microscope (Leica TCS SP2 AOBS, Leica Microsystems B.V., Rijswijk, The Netherlands). A total of five randomly selected fields at 63 times original magnification were acquired with Leica confocal software version 2.61 (Leica Microsystems, Wetzlar, Germany).

Yıldırım C., Vogel D.Y., Hollander M.R., Baggen J.M., Fontijn R.D., Nieuwenhuis S., Haverkamp A., de Vries M.R., Quax P.H., Garcia-Vallejo J.J., van der Laan A.M., Dijkstra C.D., van der Pouw Kraan T.C., van Royen N, & Horrevoets A.J. (2015). Galectin-2 Induces a Proinflammatory, Anti-Arteriogenic Phenotype in Monocytes and Macrophages. PLoS ONE, 10(4), e0124347.

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Evaluating HUVEC Adhesion and Proliferation on Substrates

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Cell attachment was evaluated by counting the cell numbers on substrates after 4-h incubation. Cell proliferation was determined by measuring the increase in cell numbers from 24 to 72 h of culture without renewal of the medium. At defined time points, the HUVECs cultured on experimental substrates were fixed with 4 % paraformaldehyde in PBS for 20 min at room temperature. Fixed cells were then permeabilized with 1 % Triton X-100 (Sigma-Aldrich) in PBS for 5 min and blocked with 1 % bovine serum albumin (BSA; Sigma-Aldrich) in PBS for 60 min. To examine the cytoskeleton, the F-actin of the fixed cells was incubated with 2 μg/mL phalloidin tetramethyl rhodamine isothiocyanate (TRITC; Sigma-Aldrich) for 60 min. The cells were also counterstained with Hoechst solution (Sigma-Aldrich) to image the nucleus. To determine the cell attachment and proliferation, the mean cell number of each substrate was analyzed from at least 10 fields at ×40 magnifications. Hoechst-stained nucleus was counted by using the “Analyze particles” tool in ImageJ software.

Chen H., Tang N., Chen M, & Chen D. (2016). Endothelialization of TiO2 Nanorods Coated with Ultrathin Amorphous Carbon Films. Nanoscale Research Letters, 11, 145.

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