DPT was obtained from the Medicinal and Chemical Institute, China Pharmaceutical University. Y27632, taxol, compound C, STO-609 were purchased from Sigma-Aldrich (St. Louis, USA). Anti-CD31, RhoA, MLC, p-MLC (Thr18/Ser19), coffilin, p-coffilin (Ser3), AMPK, p-AMPK (Thr172), LKB1, p-LKB1 (Ser428), CaMKKβ, p-CaMKKβ (Thr286) antibodies were purchased from Cell Signaling Technology (Beverly, MA). Anti-β-actin and α-tubulin antibody were purchased from Santa Cruz Biotechnology (CA, USA). Acti-stain555 Fluorescent Phalloidin was purchased from Cytoskeleton (Denver, USA).
Acti stain 555 fluorescent phalloidin
Manufactured by Cytoskeleton
Sourced in United States
Acti-stain™ 555 Fluorescent Phalloidin is a reagent used for the fluorescent labeling of F-actin in fixed and permeabilized cells. It is a high-affinity, stable probe that binds to F-actin and can be detected using a fluorescence microscope or flow cytometer.
Automatically generated - may contain errors
Lab products found in correlation
4 6 diamidine 2 phenylindole dihydrochloride dapi, by Merck Group (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Triton x 100, by Merck Group (2 mentions)
Inverted microscope, by Zeiss (2 mentions)
Anti β actin, by Santa Cruz Biotechnology (1 mentions)
Sto 609, by Merck Group (1 mentions)
Y 27632, by Merck Group (1 mentions)
α tubulin antibody, by Santa Cruz Biotechnology (1 mentions)
Penicillin, by Thermo Fisher Scientific (1 mentions)
Hank s balanced salt solution hbss, by Thermo Fisher Scientific (1 mentions)
Pluronic f 127, by Thermo Fisher Scientific (1 mentions)
Soluble mouse recombinant m csf, by R&D Systems (1 mentions)
Trap staining solution, by Merck Group (1 mentions)
Membrane dye dii, by Thermo Fisher Scientific (1 mentions)
Celltracker green, by Thermo Fisher Scientific (1 mentions)
Fluo 4 am, by Thermo Fisher Scientific (1 mentions)
Cell counting kit 8 cck 8, by Dojindo Laboratories (1 mentions)
α mem, by Thermo Fisher Scientific (1 mentions)
Rankl, by R&D Systems (1 mentions)
Normal donkey serum (nds), by Jackson ImmunoResearch (1 mentions)
12 protocols using acti stain 555 fluorescent phalloidin
1
Endothelial Cell Contractility Regulation
2
Osteoclast Differentiation Assay
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Sitagliptin was purchased from Meilun Biotech (Dalian, China) and dissolved in normal saline. Soluble mouse recombinant M-CSF and RANKL were obtained from R&D Systems (Minneapolis, MN, United States). Fetal bovine serum (FBS), alpha-MEM and penicillin were purchased from Gibco BRL (Gaithersburg, MD, United States). Triton X-100, 4′,6-diamidine-2′-phenylindole dihydrochloride (DAPI) and TRAP staining solution were obtained from Sigma–Aldrich (St. Louis, MO, United States). Cell Counting Kit-8 (CCK-8) was purchased from Dojindo Molecular Technology (Kumamoto, Japan). The Acti-stain 555 fluorescent phalloidin was obtained from Cytoskeleton Inc. (Denver, CO, United States). The Cell Tracker Green, Fluo-4 AM, Hank’s balanced salt solution (HBSS), membrane dye DiI, and Pluronic F-127 were obtained from Life Technologies (Carlsbad, CA, United States). Primary antibodies and secondary antibodies were obtained from Cell Signaling Technology (Danvers, MA, United States). Dichlorofluorescin diacetate (DCFDA) cellular ROS detection assay was purchased from Beyotime (Shanghai, China).
3
Immunocytochemical Analysis of Desmosomal Proteins
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Staining was performed, as previously described (6 (link)). The cells were fixed in 4% paraformaldehyde solution (Thermo Fisher Scientific Inc., Rockford, IL, USA) for 15 min, following which they were incubated with donkey serum blocking buffer [Tris-buffered saline containing 5% (w/v) normal donkey serum (Jackson ImmunoResearch, Hamburg, Germany)] for 30 min and the primary antibody for 60 min. Subsequently, the cells were incubated with donkey anti-mouse polyclonal immunoglobulin G (IgG) and/or donkey anti-rabbit polyclonal IgG (DyLight 488 and 594, respectively; Jackson ImmunoResearch) for 60 min at room temperature. The following antibodies were used: Mouse monoclonal anti-DSC2 (7G6; Invitrogen Life Technologies), rabbit polyclonal anti-E-cadherin (Santa Cruz Biotechnology, Inc.), mouse monoclonal anti-γ-catenin (Santa Cruz Biotechnology, Inc.), mouse monoclonal anti-DSG2 (10G11; Progen Biotechnik GmbH), mouse monoclonal anti-PKP2 (PP2/62, PP2/86, PP2/150, Progen Biotechnik GmbH), mouse monoclonal anti-pan-cytokeratin (Santa Cruz Biotechnology, Inc.) and Acti-stain™ 555 Fluorescent Phalloidin (Cytoskeleton Inc., Denver, CO, USA).
4
Cell Viability and Cytoskeletal Assessment
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On day 1 and day
14, cell-laden hydrogels were washed with Dulbecco’s PBS (DPBS)
for 5 min. Next, the cell-laden hydrogels were incubated with 0.3
μL/mL of Calcein-AM and 0.26 μL/mL of ethidium homodimer
for 1 h at room temperature protected from light. After 1 h, the cell-laden
hydrogels were washed three times for 5 min using DPBS. On day 14,
the cell-laden hydrogels were washed twice for 5 min with DPBS and
fixed with 4% paraformaldehyde for 45 min. Fixed cells within the
hydrogel were then stained with 140 nM of F-actin (Acti-stain 555
Fluorescent Phalloidin, Cytoskeleton, Inc.) in the presence of 1%
(v/v) bovine serum albumin (BSA) and 0.3% (v/v) Triton X100 at 4 °C
overnight. F-actin-stained cells in the hydrogel were washed three
times for 30 min with 1% (v/v) BSA and 0.3% (v/v) Triton X100, and
then counterstained with DAPI for 1 h at room temperature. The stained
cells in the hydrogel were washed with DPBS, and then imaged using
confocal microscopy (Olympus Fluoview, FV1000). For analysis, a total
of three hydrogels were imaged per condition with at least three random
images per gel.
14, cell-laden hydrogels were washed with Dulbecco’s PBS (DPBS)
for 5 min. Next, the cell-laden hydrogels were incubated with 0.3
μL/mL of Calcein-AM and 0.26 μL/mL of ethidium homodimer
for 1 h at room temperature protected from light. After 1 h, the cell-laden
hydrogels were washed three times for 5 min using DPBS. On day 14,
the cell-laden hydrogels were washed twice for 5 min with DPBS and
fixed with 4% paraformaldehyde for 45 min. Fixed cells within the
hydrogel were then stained with 140 nM of F-actin (Acti-stain 555
Fluorescent Phalloidin, Cytoskeleton, Inc.) in the presence of 1%
(v/v) bovine serum albumin (BSA) and 0.3% (v/v) Triton X100 at 4 °C
overnight. F-actin-stained cells in the hydrogel were washed three
times for 30 min with 1% (v/v) BSA and 0.3% (v/v) Triton X100, and
then counterstained with DAPI for 1 h at room temperature. The stained
cells in the hydrogel were washed with DPBS, and then imaged using
confocal microscopy (Olympus Fluoview, FV1000). For analysis, a total
of three hydrogels were imaged per condition with at least three random
images per gel.
5
Macrophage Morphology Observation Protocol
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Morphologies of treated macrophages were observed and photographed under an inverted microscope (ZEISS, German). For fluorescent observation, macrophages were fixed in paraformaldehyde and permeabilized beforehand. The cytoskeleton was labeled with Acti-stain™ 555 Fluorescent Phalloidin (Cytoskeleton, USA) and nuclei with DAPI (YEASEN, China). Fluorescently labelled cells were examined using a ZEISS fluorescent imaging microscope (ZEISS, German).
6
Osteoclast Differentiation Assay
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DAC (CAS No. 31565-50-1, MOLBASE Biotechnology Co. Ltd, Shanghai, China) was dissolved in dimethyl sulfoxide without exposure to light and stored at −20°C. Dulbecco’s modified Eagle’s medium (DMEM) was provided by Hyclone (Logan, UT, USA). Penicillin–streptomycin solution, trypsin–ethylenediaminetetraacetic acid (EDTA) solution (0.25%), and fetal bovine serum (FBS) were obtained from Gibco (Gaithersburg, MD, USA). Recombinant mouse RANKL and macrophage-colony-stimulating factor (M-CSF) were provided by PeproTech (Rocky Hill, NJ, USA). A tartrate-resistant acid phosphatase (TRAP) staining kit, Triton X-100, and 4,6-diamidine-2-phenylindole dihydrochloride (DAPI) were purchased from Sigma-Aldrich. A Cell Counting Kit-8 (CCK-8) was provided by Dojindo Molecular Technology Inc. (Kumamoto, Japan). Acti-stain 555 fluorescent phalloidin was obtained from Cyto-skeleton Inc. (Denver, CO, USA). The specific primary antibodies and secondary antibodies used in the experiments were provided by Cell Signaling Technology (Danvers, MA, USA).
7
Fibronectin-Coated Glass Coverslip Assay
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Glass coverslips were coated with 0.01% poly‐L‐lysine (P8920‐100ML, Sigma‐Aldrich), air‐dried overnight, and fixed with 0.5% glutaraldehyde. Fibronectin HiLyte 488 (FNR02‐A, Cytoskeleton, Denver, CO, USA) was added dropwise to each glass coverslip. The coverslips were then transferred to a 24‐well plate and incubated at 37 °C for 1 h. Cells were suspended following trypsinization and added to the glass coverslips. After 48 h, cells on each glass coverslip were fixed, permeabilized and labelled with Acti‐Stain™ 555 fluorescent phalloidin (PHDH1, Cytoskeleton), followed by mounting in anti‐fluorescence attenuating reagent (S2110, Solarbio, Beijing, China). For the determination of matrix degradation, the coverslips were photographed using a laser confocal microscope (ZEISS LSM 800, Carl Zeiss, Jena, Germany).
8
Immunofluorescence Staining of CXCR6 and β-Catenin
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Subconfluent cells grown on glass coverslips are fixed with 3.7% parafolmaldeide in PBS for 10min, permealized with 0.5% Triton X-100 in PBS for 5min at room temperature and incubated with 10% goat serum in PBS for 1hr. The cells are stained with polyclonal anti-CXCR6 antibody (1:400, Abcam, Ab8023) or anti-β-catenin (MIllipore, MAB2081, 1:200 nostro; anti-Active βCatenin (Anti-ABC) Antibody, Millipore, 05-665, 1:50) or Anti-Fascin antibody (DyLight 488, ab156578 Abcam) overnight at 4 °C. Thus, after a brief washing with PBS, CXCR6 staining cells are incubated with the secondary antibody (anti rabbit Alexa488 1:250) for 1 h. The cytoskeleton is stained with Falloidin (Acti-stain 555 Fluorescent Phalloidin, Cytoskeleton Inc, 100 nM) at room temperature for 45 min. The nuclei are counterstained with DAPI and the slides mounted with Pro-long anti fade reagent (Life technologies). The images are acquired with a Leika TCS NT confocal microscope.
9
Visualizing GPCMV Infection by DiO Labeling
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DiO (Vybrant cell-labeling solution, Molecular Probe) was added to a GPCMV WT stock (4.1 × 107 PFUs in 180 μl of PBS) that was partially purified by sucrose gradient centrifugation15 (link), at a final concentration 2 μM, and the mixture was incubated for 1 hr at room temperature. The mixture was used as the DiO-labeled GPCMV stock. GPE-7 cells in 8-well chamber slides (Nunc) were incubated with the DiO-labeled GPCMV at an MOI of 22.5 for 1 hr at 4 °C, and then the inoculum was replaced with warmed culture medium. The cells were incubated for the indicated duration at 37 °C, fixed with 3.7% formalin, stained with DAPI (NucBlue Fixed Cell Stain Ready Probes reagent, Life Technologies) for detection of nuclei and with 300-fold diluted acti-stain 555 fluorescent phalloidin (Cytoskeleton, Inc.) for detection of the polymerized form of F-actin, and observed under a confocal microscope. Images of 10 fields (0.17μm2/field) for each set of conditions were captured, and the virus signals were counted.
10
Visualizing MSC Cytoskeleton Structure
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Cytoskeleton of MSC was stained with Acti-stain 555 Fluorescent Phalloidin (Cytoskeleton, CO, USA, 1:500) according to the manufacturer’s instructions.
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