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Flash phalloidin green 488

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Flash Phalloidin Green 488 is a fluorescent dye used to label filamentous actin (F-actin) in cells. It binds to F-actin with high affinity, allowing for the visualization and analysis of the actin cytoskeleton.

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Lab products found in correlation

Blue fluorescent hoechst, by Thermo Fisher Scientific (2 mentions) A1r hd upright multiphoton confocal microscope, by Nikon (2 mentions) 4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions) Goat serum, by Thermo Fisher Scientific (1 mentions) Eclipse, by Nikon (1 mentions) Tween 20, by Merck Group (1 mentions) 4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions) Ix83 fluorescence microscope, by Olympus (1 mentions) H2o2 treatment, by Merck Group (1 mentions) Calcein am, by Thermo Fisher Scientific (1 mentions) A1rs hd confocal microscope, by Nikon (1 mentions) Objective len, by Olympus (1 mentions) 4 6 diamidino 2 phenylindole (dapi), by Nacalai Tesque (1 mentions) Fv1200 laser confocal microscope, by Olympus (1 mentions) 8 well chamber slide, by Merck Group (1 mentions) Duolink image tool software, by Merck Group (1 mentions) Axioimager z1 apotome, by Zeiss (1 mentions) Bovine serum albumin (bsa), by Merck Group (1 mentions) Saponin, by Merck Group (1 mentions) Vectashield antifade mounting medium, by Vector Laboratories (1 mentions)

10 protocols using flash phalloidin green 488

1

Fluorescent Labeling of Tendon-Derived Stem Cells

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TDSCs on normal and degenerative DTSs were fixed with 4% paraformaldehyde at room temperature for 15 minutes, followed by washing with PBS 3 times, then treated with PBS containing 0.1% Tween-20 (Sigma) and 1% goat serum (Thermo) for 15 minutes, and washed with PBS 3 times. Finally, the TDSs were incubated with Flash Phalloidin Green 488 (1 : 20) (BioLegend) and DAPI (Sigma) at 4°C in the dark for 30 minutes. Photos were taken by an inverted fluorescence microscope (Eclipse, Nikon).
Liu C., Luo J.W., Zhang K.K., Lin L.X., Liang T., Luo Z.P., Zhuang Y.Q, & Sun Y.L. (2018). Tendon-Derived Stem Cell Differentiation in the Degenerative Tendon Microenvironment. Stem Cells International, 2018, 2613821.
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2

Cytoskeleton and Nucleus Staining of Human Fibroblasts

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After human fibroblast cells [32 (link)] were cultured on the top of the working electrode film in the culture plate, the cytoskeleton and nucleus were stained with Flash Phalloidin Green 488 (BioLegend) and blue fluorescent Hoechst (352/461 nm) (Thermo Fisher Scientific), respectively. The Flash Phalloidin Green 488 was reconstituted with 1.5 ml of methanol to make 300-unit stock solution. The samples were fixed with 2–4% formaldehyde for 15 min and then rinsed three times with prewarmed PBS. The samples were incubated with Flash Phalloidin Green 488 (diluting 300 µl stock solution 1:50 in 1× PBS) and Hoechst (1 µg ml−1) for 30 min at 37 °C. After staining, the cells were rinsed with prewarmed buffer for three times and imaged samples using a Nikon A1RS high definition (HD) Confocal microscope.
Liu A., Long Y., Li J., Gu L., Karim A., Wang X, & Gibson A.L. (2021). Accelerated complete human skin architecture restoration after wounding by nanogenerator-driven electrostimulation. Journal of Nanobiotechnology, 19, 280.
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3

Phagocytosis of Apoptotic Cells by BMDMs

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BMDMs were plated at 5×104 cells/well in 8-well glass slides. The macrophages were labeled with cell-permeant dye Calcein, AM (Cat# C1430, Thermo Fisher Scientific, USA) according to the manufacturer’s instructions for 2 h. Apoptosis in H9c2 cells was induced by hydrogen peroxide (H2O2) treatment (Sigma-Aldrich, USA) for 4 h. The apoptotic cells were labeled with PKH26 (red fluorescence, Catalog-MINI26-1KT, Sigma-Aldrich, USA) according to the manufacturer’s instructions. Apoptotic H9c2 cells were overlaid on BMDMs (1:1 ratio of macrophage: apoptotic cells) and incubated for 2 h. After 2 h, cells were washed 4 times with ice-cold PBS to remove the unengulfed apoptotic cells. The cells were fixed with 4% paraformaldehyde, counterstained with DAPI (Thermo Fisher Scientific, USA), and mounted for fluorescence microscopy (Olympus IX83 fluorescence microscope). Phagocytosis was determined by counting cells containing engulfed red fluorescent apoptotic cells. A minimum of 300 macrophages was counted per well in triplicate. Data were represented as percent phagocytosis, i.e., the total number of cells with ingested apoptotic cells divided by the total number of macrophages counted times 100. In a separate phagocytosis experiment, BMDMs were stained with Flash Phalloidin™ Green 488 (Cat# 424201, BioLegend, USA) to observe macrophage cell morphology.
Singh S., Henderson J.C., Patil M., Dubey P.K., Dubey S., Kannappan R., Zhang J, & Krishnamurthy P. (2022). MicroRNA-181c-5p modulates phagocytosis efficiency in bone marrow-derived macrophages. Inflammation research : official journal of the European Histamine Research Society ... [et al.], 71(3), 321-330.
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4

3D Printed Film Cell Morphology Analysis

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After 3T3 cells were cultured on the 3D printed film with different polarities or cell plates (control group) in 24-well plates, the cell morphology was observed directly using an upright confocal microscope (Nikon A1R high definition (HD) Upright Multiphoton/Confocal microscope). The cytoskeleton and nucleus were stained with Flash Phalloidin Green 488 (BioLegend) and blue fluorescent Hoechst (352/461 nm) (Thermo Fisher Scientific), respectively. Reconstitute the Flash Phalloidin Green 488 with 1.5 mL of methanol to make 300 units stock solution. The samples were fixed with 2–4% formaldehyde for 15 min and then rinsed three times with prewarmed PBS. The samples were incubated with Flash Phalloidin Green 488 (diluting 300 mL stock solution 1:50 in 1X PBS) and Hoechst (50 × 10−9m) for 30 min at 37 °C. After staining, the cells were rinsed with prewarmed buffer for three times and imaged samples using a Nikon A1R HD Upright Multiphoton/Confocal microscope.
Li J., Long Y., Yang F., Wei H., Zhang Z., Wang Y., Wang J., Li C., Carlos C., Dong Y., Wu Y., Cai W, & Wang X. (2020). Multifunctional Artificial Artery from Direct 3D Printing with Built-In Ferroelectricity and Tissue-Matching Modulus for Real-Time Sensing and Occlusion Monitoring. Advanced functional materials, 30(39), 2002868.
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5

3T3 Cell Cytoskeleton Imaging Protocol

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After 3T3 cells were cultured on the films (PVDF, PDMS, Parylene-C) or in 24-well plates (control group), the cell morphology was observed directly using a confocal microscope (Nikon A1RS HD Confocal Microscope). The cytoskeleton and nucleus were stained with Flash Phalloidin™ Green 488 (BioLegend) and blue fluorescent Hoechst (352/461 nm) (Thermo Fisher Scientific), respectively. Reconstitute the Flash Phalloidin™ Green 488 with 1.5 ml of methanol to make 300 units stock solution. The samples were fixed with 2–4% formaldehyde for 15 min and then rinsed three times with prewarmed phosphate-buffered saline (PBS). The samples were incubated with Flash Phalloidin™ Green 488 (diluting 300 μL stock solution 1:50 in 1X PBS) and Hoechst (50 nM) for 30 min at 37 °C. After staining, the cells were rinsed with prewarmed buffer three times and samples imaged using a Nikon A1R HD Upright Multi-Photon/Confocal microscope.
Li J., Hacker T.A., Wei H., Long Y., Yang F., Ni D., Rodgers A., Cai W, & Wang X. (2021). Long-term in vivo operation of implanted cardiac nanogenerators in swine. Nano energy, 90(Pt A), 106507.
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6

Neutrophil Polarization Assay

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Neutrophil polarization assay was performed using neutrophils purified with kits as described above. The purified neutrophils were serum-starved in RPMI-1640/0.1% BSA for 4 h, treated with or without arbutin for 30 min at 37°C in an 8-well chamber slide (Merck, CA, USA), and then stimulated with CXCL2 for 2 min at 37°C. The cells were fixed with 4% PFA (Wako pure Chemica) for 10 min, permeabilized with 0.1% Triton-X for 10 min, and then stained with Flash Phalloidin Green 488 (BioLegend) for 20 min at room temperature. The nucleus was stained with DAPI (Nacalai-Tesque). The images were randomly acquired using an FV1200 confocal laser microscope with a 40x objective lens (Olympus, Tokyo, Japan). The number of total neutrophils was counted with Duolink Image Tool software (Sigma). Polarized neutrophils were determinized by nonuniform phalloidin staining (F-actin).
Kobayashi D., Watarai T., Ozawa M., Kanda Y., Saika F., Kiguchi N., Takeuchi A., Ikawa M., Matsuzaki S, & Katakai T. (2022). Tas2R signaling enhances mouse neutrophil migration via a ROCK-dependent pathway. Frontiers in Immunology, 13, 973880.
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7

Immunofluorescence Staining Protocol for U87 Cells

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U87 cells (grown for 48 h on glass slides or following cytospin at 400 rpm for 3 min) were fixed for 30 min in 4% paraformaldehyde in PBS. For actin staining, cells were permeabilized and blocked with 0.1% saponin (Sigma) and 0.1% BSA (MD Millipore-Merck, Burlington, MA, USA) in PBS–Tween (PBS-T) and incubated for 1 h with Flash Phalloidin Green 488 (1:100; BioLegend, San Diego, CA, USA) in the dark [16 (link)]. For LRP1B expression analysis, cells were permeabilized with 0.5% Triton X-100 in PBS, blocked with 3% BSA and 0.1% Triton X in PBS, and incubated with LRP1B antibody (1:500 in blocking buffer; Sigma SAB4200326) overnight at 4 °C. After washing with PBS, slides were further incubated with secondary anti-rabbit antibody (Alexa 594, Invitrogen A-11037) for 1h at room temperature. All slides were incubated with DAPI (1:1000) for 5 min, washed, and mounted with Vectashield antifade mounting medium (Vector Laboratories). Fluorescence was analyzed using LEICA DM 2000 or with Zeiss Axio Imager Z1 Apotome microscopes with a coupled camera. Nuclear morphometric analysis (NMA) was carried out in images from DAPI-stained nuclei (with at least 300 dpi) using ImageJ analysis software with NII Plugin [82 (link)].
Peixoto J., Príncipe C., Pestana A., Osório H., Pinto M.T., Prazeres H., Soares P, & Lima R.T. (2023). Using a Dual CRISPR/Cas9 Approach to Gain Insight into the Role of LRP1B in Glioblastoma. International Journal of Molecular Sciences, 24(14), 11285.
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8

Visualizing Actin Cytoskeleton with Fluorescent Probes

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Cells were grown in sterilized round slides and transfected with siRNAs, as previously described, before being stained with “Flash Phalloidin™ Green 488” (Biolegend, San Diego, CA, USA) (1:200) and DAPI (1:1000) as previously described [49 (link)]. Samples were photographed using the fluorescence microscope Zeiss Axio Imager Z1 (Carl Zeiss, Oberkochen, Germany). Data was analyzed using Image J Version 1.53t software (National Institutes of Health, Bethesda, MD, USA).
Teixeira E., Fernandes C., Bungărdean M., Paula A.D., Lima R.T., Batista R., Vinagre J., Sobrinho-Simões M., Máximo V, & Soares P. (2024). Investigating USP42 Mutation as Underlying Cause of Familial Non-Medullary Thyroid Carcinoma. International Journal of Molecular Sciences, 25(3), 1522.
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9

Efferocytosis Assay for Macrophages

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The in vitro efferocytosis studies were performed using both BMMØs and PMØs, and apoptotic SNSCs as previously described with slight modifications [19 ]. Briefly, macrophages were seeded into four-well slides at 2 × 104 cells per well and incubated overnight in the humidified chamber at 37 °C and 5% CO2. Macrophages were treated with either LPS (50 ng/mL), or LPS (50 ng/mL) + EPO (5 IU/mL), or not treated (control group) for 24 h and then incubated with PKH26 (cell membrane labeling dye; # MINI26; Sigma-Aldrich) labeled apoptotic SNSCs for 3 h (ratio: 1 macrophage:2 SNCSs). After incubation, macrophages were washed (1XDPBS) and labeled with Flash Phalloidin Green 488 (1:100; #42420; Biolegend) for intracellular cytoskeleton F-actin and coverslips were mounted on glass slides with ProLongTM Gold anti-fade reagent with DAPI for examination under a fluorescent microscope. Efferocytosis percentages were calculated using NIH-ImageJ Version 1.53 software (USA) by analyzing the ratio of PKH26 to DAPI.
Govindappa P.K, & Elfar J.C. (2022). Erythropoietin promotes M2 macrophage phagocytosis of Schwann cells in peripheral nerve injury. Cell Death & Disease, 13(3), 245.
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10

Immunofluorescence Staining of Actin

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Cells were fixed in 4% PFA for 10 min at room temperature at the end of treatment, permeabilized for 10 min with 0.5% Triton X-100 and washed twice with PBS. The cells were blocked for 30 min with 5% FBS at room temperature. Cells were then stained with Flash Phalloidin Green 488 (cat# 42420, Biolegend, CA, USA). At the end, coverslips mounted on glass slides with VECTASHIELD antifade mounting medium with 4’, 6-diamidino-2-phenylindole-DAPI (cat# H-1200, Vector Laboratories, CA, USA) and observed under inverted microscope (IX83, Olympus, TYO, JP). Images were analyzed for cell area measurements using NIH’s image J software.
Narasimhan G., Henderson J., Luong H.T., Rajasekaran N.S., Qin G., Zhang J, & Krishnamurthy P. (2019). OBG-like ATPase 1 inhibition attenuates angiotensin II-induced hypertrophic response in human ventricular myocytes via GSK-3beta/beta-catenin signaling. Clinical and experimental pharmacology & physiology, 46(8), 743-751.
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