Astrocytes were cultured on poly-L-lysine-coated coverslips and serum-starved DMEM for 24 h. The cells were then treated with either isoflavones or E2 for 30 min then washed with PBS and fixed with 4% PFA followed by blocking with 2% FBS. The cells were incubated with CytoPainter Phalloidin-iFluor 594 reagent (Abcam, Cambridge, UK) and nuclei were stained with DAPI and then visualized under a laser confocal scanning microscope (Zeiss LSM 880, Carl Zeiss Microscopy GmbH). The degree of cytoskeletal rearrangement was examined using the FiloQuant by ImageJ Fiji (NIH) or cortical F-actin score CFS index (31 (link)). The CFS index was determined based on at least three independent experiments. Briefly, F-actin cytoskeletal reorganization for each cell was scored on a scale ranging from 0 to 3, based on the degree of cortical F- actin ring formations 0, no cortical F-actin, normal stress fibers; 1, cortical F-actin deposits below half the cell border; 2, cortical F-actin deposits exceeding half the cell border; and 3, complete cortical ring formatting and/or total absence of central stress fiber. A minimum of 50 cells were examined from each group in each independent experiment, and the CFS index for treated astrocytes was the average score of the counted cells ± standard error of the mean (SEM).
Cytopainter phalloidin ifluor 594 reagent
Manufactured by Abcam
Sourced in United States
CytoPainter Phalloidin-iFluor 594 Reagent is a fluorescent probe used to label and visualize actin filaments in cells. It binds specifically to F-actin, allowing for the detection and analysis of the cytoskeleton structure.
Automatically generated - may contain errors
Lab products found in correlation
Lsm 880, by Zeiss (3 mentions)
Alexa fluor 488 conjugate, by Thermo Fisher Scientific (1 mentions)
Anti phospho rac1 cdc42 ser71, by Cell Signaling Technology (1 mentions)
Anti phospho akt ser473 d9e xp, by Cell Signaling Technology (1 mentions)
Anti phospho p44 42 mapk erk1 2 thr202 tyr204, by Cell Signaling Technology (1 mentions)
Sb431542, by Merck Group (1 mentions)
Protease and phosphatase inhibitor, by Merck Group (1 mentions)
Human tgf β1, by R&D Systems (1 mentions)
Pd98059, by Cell Signaling Technology (1 mentions)
8 well chamber slide, by Corning (1 mentions)
Las x, by Leica (1 mentions)
Evos xl core cell imaging system, by Thermo Fisher Scientific (1 mentions)
Sp8 confocal microscope, by Leica (1 mentions)
Vectasheild with dapi, by Vector Laboratories (1 mentions)
Mouse monoclonal anti β tubulin 3, by Merck Group (1 mentions)
Triton x 100, by Merck Group (1 mentions)
Paraformaldehyde (pfa), by Fujifilm (1 mentions)
Dakocytomation fluorescent mounting medium, by Agilent Technologies (1 mentions)
4 6 diamidino 2 phenylindole dapi, by Fujifilm (1 mentions)
Bz x700 digital microscope, by Keyence (1 mentions)
7 protocols using cytopainter phalloidin ifluor 594 reagent
1
Quantifying Astrocyte Cytoskeletal Reorganization
2
Isoflavone and E2 Signaling Pathways
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Cultured cells were exposed to isoflavones or E2 for 30 min then rinsed three times with PBS, fixed with 4% PFA, and blocked with 2% FBS. Cells were then incubated with rabbit monoclonal anti-phospho-Akt (Ser473) (D9E) XP (1:200; Cell Signaling, MA, USA), anti-phospho-p44/42 MAPK (ERK1/2) (Thr202/Tyr204) (1:200; Cell Signaling), or anti-phospho-Rac1/Cdc42 (Ser71) (1:200; Cell Signaling) antibodies, followed by CytoPainter Phalloidin-iFluor 594 reagent (Abcam) and donkey anti-rabbit IgG (H+L) secondary antibodies, Alexa Fluor® 488 conjugate (1:200; Thermo Fisher Scientific, Inc, Waltham, MA, USA). Cell nuclei were also stained with DAPI. The cells were then inspected using a laser confocal scanning microscope (Zeiss LSM 880, Carl Zeiss Microscopy GmbH).
3
TGF-β1 Signaling Pathway Modulation
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Human TGF-β1 (R&D Systems), protease and phosphatase inhibitors (Sigma-Aldrich), PD98059 (Cell Signaling Technology), SB431542 (Sigma). CytoPainter Phalloidin-iFluor 594 Reagent (Abcam) was used for phalloidin staining.
4
Visualizing Uropathogenic E. coli Interaction with Urothelial Cells
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RT4 cells were seeded onto 8‐well chamber slides (Corning) at 3 × 104 cells per well and grown to confluence. Cells either preconditioned or not with HA (2 mg mL−1) were challenged for up to 24 h with TPA4935J, a clinical UPEC isolate transformed with pEGFp‐C3 (102 per well). After washing with PBS, fixing in 4% PFA the cells were stained for 2 h with CytoPainter Phalloidin‐iFluor 594 reagent (Abcam) diluted 1:1000, and coverslips mounted using Vectasheild with DAPI (Vector Laboratories). Images were captured using a Leica SP8 confocal microscope (fluorescence) or an EVOS XL Core Cell Imaging System (Thermo Fisher; light microscope images). Image analysis was conducted using LAS X (Leica) and Image J software.
5
Thyroid Hormone Effects on Neurite Outgrowth
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Cultured cells were exposed to 10 nM of T3, T4, or rT3 (Sigma) for one to three days. Cells were washed with PBS three times, then fixed with 4% PFA, and continued with blocking with 5% FBS in PBS. Neuro-2A cells were immunostained with neuronal and presynaptic antibodies, mouse monoclonal anti-β-tubulin III (1:200) (Sigma) and rabbit anti-syn-1 (1:200) (Cell Signaling, MA, USA), continued with secondary antibody donkey anti-mouse Alexa Fluor® 405 (1:200), donkey anti–rabbit Alexa Fluor® 488 conjugate (1:200) (Thermo Fisher Scientific, Inc.), and CytoPainter Phalloidin–iFluor 594 reagent (Abcam). Neuro-2A cells were observed under a laser confocal scanning microscope (Zeiss LSM 880, Carl Zeiss Microscopy GmbH). Filopodia were counted manually. Data are showed as the mean ± standard error of the mean (SEM). More than three independent experiments were performed. Results were consistent between each experiment. A representative result from one experiment is shown.
6
Immunofluorescence Staining of Cells
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Cells were fixed with 4% paraformaldehyde (Wako Pure Chemical Industries, Ltd., Saitama, Japan) for 10 min at 24°C, and reacted with 0.1% TritonX-100 (Sigma-Aldrich) and 5% of goat normal serum (Agilent Technologies) in PBS (Wako Pure Chemical Industries, Ltd.) for 10 min. Cells were then incubated overnight with each primary antibody (S6 Table ) in PBS at 4°C. They were then incubated at 24°C with the secondary antibody for each primary antibody conjugated with Alexa Fluorescent dye (1:300 dilution, Thermo Fisher Scientific Inc.). The nuclei were counterstained with 4, 6-diamidino-2-phenylindole (DAPI) (Wako Pure Chemical Industries, Ltd.) for 45 min. To prevent fading, cells were then mounted in DakoCytomation Fluorescent Mounting Medium (Agilent Technologies). Samples were observed and images were captured with an Olympus IX71 inverted microscope (Tokyo, Japan) or a Keyence BZ-X700 digital microscope (Osaka, Japan). F-actin was discerned by staining with CytoPainter Phalloidin-iFluor 594 Reagent (ab176757, Abcam).
7
Immunofluorescence Staining of Cardiac Fibroblasts
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Freshly isolated adult rat cardiac fibroblasts were plated on acid-washed 18 mm glass coverslips in 12-well plates, then treated with CB-839 and/or TGFβ1 as above. Cells were fixed with 4% paraformaldehyde for 30 min at room temperature followed by 3 washes in PBS, then permeabilized in 0.1% Triton X-100 in PBS for 5 min followed by 3 washes in PBS. Coverslips were incubated for 1 h at room temperature with CytoPainter Phalloidin-iFluor 594 Reagent (ab176757; Abcam, Canada) as per the manufacturer’s instructions. Staining solution was removed, and coverslips were washed 3 times with PBS. Coverslips were mounted onto slides using ProLong Gold Antifade Mountant with DAPI (ThermoFisher Scientific, Canada). Cells were imaged on a Nikon Eclipse TE2000S fluorescent microscope using a 20× objective. NIS software (Nikon, Melville, NY, USA) was used for image capture, and Image J (NIH, USA) was used to merge captured images.
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