Inhibition experiments were performed with blebbistatin (Sigma-Aldrich, USA) or with the ROCK inhibitor Y27632 (Sigma-Aldrich, USA) at concentrations as indicated. Cells were fixed with 2% paraformaldehyde (Sigma-Aldrich, USA) in PBS. Actin cytoskeleton was visualized with phalloidin-Alexa Fluor 647 (ThermoFisher, USA). For flow cytometry, integrin β6 was stained as described previously with an antibody developed by the antibody facility at the University of Geneva (Kessler et al., 2022 (link)). Integrin αV was stained with a commercial antibody (ThermoFisher, USA, clone RMV-7, 1:1000 diluted). Labeling of primary antibodies was visualized with anti-mouse PE and anti-rat PE (both Southern Biotech, USA, 1:1000 diluted). Actin in living cells was visualized with SiR actin (Spirochrome, Switzerland).
Sir actin
Manufactured by Spirochrome
Sourced in Switzerland
SiR-actin is a fluorescent probe for labeling actin in live cells. It is a silicon-rhodamine (SiR) derivative that binds specifically to actin filaments. SiR-actin can be used to visualize the dynamics of the actin cytoskeleton in living cells.
Automatically generated - may contain errors
Lab products found in correlation
Paraformaldehyde (pfa), by Merck Group (3 mentions)
Blebbistatin, by Merck Group (2 mentions)
Alexa fluor 647 phalloidin, by Thermo Fisher Scientific (2 mentions)
Hoechst 33342, by Thermo Fisher Scientific (2 mentions)
Sir tubulin, by Spirochrome (2 mentions)
Tcs sp8 confocal microscope, by Leica (2 mentions)
Sir700 dna, by Spirochrome (2 mentions)
Alexa fluor 568 phalloidin, by Thermo Fisher Scientific (2 mentions)
Y 27632, by Merck Group (1 mentions)
Eclipse ti e microscope, by Nikon (1 mentions)
Saponin, by Merck Group (1 mentions)
Lsm710 upright confocal microscope, by Zeiss (1 mentions)
Hank s balanced salt solution, by Thermo Fisher Scientific (1 mentions)
Syto 16, by Thermo Fisher Scientific (1 mentions)
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (1 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
Hepes, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 568, by Thermo Fisher Scientific (1 mentions)
Mitotracker deep red fm, by Thermo Fisher Scientific (1 mentions)
Gfp booster atto488, by Proteintech (1 mentions)
33 protocols using sir actin
1
Cytoskeleton Dynamics Modulation by Inhibitors
2
Fluorescence Immunostaining of Myosin Heavy Chain
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For fluorescence immunostaining of myosin heavy chain, cells were fixed with 4% paraformaldehyde (Sigma) in PBS for 10min. Fixation time for 3D protrusions was set to 1h. Cells were subsequently permeabilized for 30min with 0.1% Saponin (Sigma) while blocked with 0.1% bovine serum albumin (BSA, Sigma). Finally, fixed cells were incubated with primary antibodies. Myosin-4 Monoclonal Antibody conjugated with Alexa Fluor 488 (MF20, Thermofisher) was incubated for 1h at room temperature, at 5μg/mL (1:100 dilution) and 0.1% BSA.
Actin was labelled with SiR-actin (Spirochrome). Concentrations used were 1μM (30min incubation) for fixed samples and 100nM (6h incubation) for live imaging.
Cell nuclei were labelled after fixation with Hoechst 33342 (Thermofischer) at 10μg/mL (5min incubation).
Fixed samples were imaged by using a Nikon Eclipse Ti-E microscope equipped with a Nikon A1 confocal unit. We employed x40/x60 water/oil immersion objectives (NA 1.15/1.4). The microscope was operated with NIS-Elements (ver. 4.60.00). A Zeiss LSM-710 upright confocal microscope (40x objective, NA 0.75) was used for imaging the top and exterior of cellular protrusions. The microscope was operated with Zeiss Zen 2011.
Actin was labelled with SiR-actin (Spirochrome). Concentrations used were 1μM (30min incubation) for fixed samples and 100nM (6h incubation) for live imaging.
Cell nuclei were labelled after fixation with Hoechst 33342 (Thermofischer) at 10μg/mL (5min incubation).
Fixed samples were imaged by using a Nikon Eclipse Ti-E microscope equipped with a Nikon A1 confocal unit. We employed x40/x60 water/oil immersion objectives (NA 1.15/1.4). The microscope was operated with NIS-Elements (ver. 4.60.00). A Zeiss LSM-710 upright confocal microscope (40x objective, NA 0.75) was used for imaging the top and exterior of cellular protrusions. The microscope was operated with Zeiss Zen 2011.
3
Live Imaging of Embryonic Cortical Neurons
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Time-pregnant mice were deeply anesthetized with 10% chloral hydrate. Brains of the embryos were dissected in ice-cold PBS (Invitrogen) supplemented with 0.65% glucose. The hemispheres were separated and incubated with SYTO16 (1:1000; Invitrogen) and SiR-actin (1:1000; Spirochrome) in DMEM (Invitrogen) for 20 min on ice. After transferring the hemispheres to ice-cold DMEM, the somatosensory cortex was dissected and placed into glass-bottom dishes (Matec) that were coated with poly-l -lysine (1 μg/ml) and recombinant proteins in different combinations (10 ng each) for 1 hour at 37°C. After incubation for 20 min at RT in 100 μl of culture medium [DMEM (Invitrogen) with 25% Hanks’ balanced salt solution (Invitrogen), 10% FBS, penicillin (10,000 U/ml), streptomycin (10,000 μg/ml), 0.65% d -glucose, and 0.4 mM l -glutamine] supplemented with 0.5% methyl cellulose, compartments were filled with culture medium and 0.1 mM Hepes (Invitrogen) before live cell imaging was performed. Pictures were taken using IQ3 software with multi-positions and Z-stack protocols. To reduce exposure time and laser intensity, acquisitions were done using binning 1 × 1.
4
Live F-actin Imaging Protocol
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The live F-actin dataset was acquired for this study and was used to train: (1) the TA-CycleGAN for live and fixed domain adaptation and (2) the TA-GANLive. The live F-actin dataset consists of 800 paired STED and confocal images of F-actin stained with the fluorogenic dye SiR-actin (Spirochrome) in living hippocampal cultured neurons (Supplementary Table 1 ). The dataset was split into a training set (753 images) and a validation set (47 images). The images were of variable size (from a minimum width of 2.76 to a maximum of 49.1 μm, pixel size is always 20 nm).
5
Multicolor Fluorescent Labeling Protocol
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Primary antibodies anti-EEA1 (ab70521), anti-58K Golgi (ab27043), antipericentrin (ab28144), and anticatalase (ab110292) were purchased from Abcam, anti-TGN46-8 (SAB4200355) from Sigma and anti-Tom20 (sc-17764) from Santa Cruz Biotechnology. All primary antibodies were monoclonal mouse antibodies. Polyclonal secondary antimouse antibodies conjugated to Atto647N (50185, Sigma Aldrich) as well as isotype-specific secondary antimouse antibodies conjugated to Atto647N (610-156-041, Rockland Immunochemicals) and Alexa Fluor 568 (A-21124, Thermo Fisher Scientific) were used for detection. The nanoboosters—single-domain alpaca antibody fragments covalently coupled to fluorescent dyes—GFP-Booster Atto488 and RFP-Booster Atto594 (Chromotek) were used to boost eYFP and mCherry fluorescence, respectively. Live cell stains SiR-tubulin, SiR-actin, and SiR-lysosome were from Spirochrome and MitoTracker Deep Red FM was from Thermo Fisher Scientific.
6
Fluorescent Labeling of Actin and Synaptic Vesicles
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Neurons at 10 div were incubated for 1 h at 37°C, 5% CO2 in NB+ medium containing 100 nM SiR-actin (Spirochrome). Neurons were then incubated in an HBS medium for 10 min at 37°C. Following this incubation, the neurons were transferred to a live-cell imaging chamber (Life Imaging Services) containing HBS medium and placed at 37°C in the incubated chamber of an epifluorescence/TIRF microscope (Zeiss). HBS medium was then exchanged for HBS/KCL medium containing APV at 50 µM, CNQX at 25 µM, and FM1-43 (SynaptoGreen-C4; Biotium) at 5 µM for 180 s at 37°C. HBS/KCL medium was then changed back to HBS medium after three 1-min 30-s rinses with HBS medium. Neurons were then left to recover for 15 min in an HBS medium. Green (FM1-43) and far-red (SiR-actin) channel epifluorescence images were taken using a 40X objective for several fields of view selected for the presence of a number of beads induced. FM1-43 release was achieved following an additional 3-min incubation with HBS/KCl medium with APV/CNQX, followed by three rinses with HBS medium every 1 min 30 s, and 15 min recovery in HBS medium. After release, images of the same preselected fields of view were then acquired using the same settings as the post-loading images.
7
Morphological Response of MuSCs to Fluid Shear Stress
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MuSCs were seeded at 4–17 × 103/cm2 on matrigel (Corning)-coated glass slides (22 × 22 mm) and cultured for 1–5 days. Three to 5 h before fluid shear stress application, culture medium was refreshed by medium with low serum (2% FBS) containing 250 nM live cell stains for F-actin (SiR-Actin, Spirochrome, Stein am Rhein, Switzerland) and nucleic acid (SiR700-DNA, Spirochrome). To determine the effect of fluid shear stress on MuSC morphology, the glass slide was placed into a microfluidic chamber connected to a pump. MuSCs were then subjected to CFSS for 2 min by pumping 7 ml medium with a shear stress of 1 Pa/s, and imaged using Leica TCS SP8 confocal microscope as described [20 (link)]. Cells were irradiated with a pulsed white light laser at a wavelength of 645 nm, and XZ time-lapse images were acquired using 40× 1.3 NA oil objective. The effect of CFSS on cell apex-height was quantified during the initial 5 sec of CFSS application.
8
Assessing Cell Proliferation and Actin Dynamics
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Proliferation was assessed by the evaluation of Ki-67 positive staining. Briefly, 100,000 cells were seeded in P24 well plates and grown until fully confluent (circa 24 h). A wound was performed by scratching with a pipette tip, and cells were grown for 24 h. Then, cells were fixed with PFA for 20 min and left overnight with PBS at 4 °C. Cells were then permeabilized with 0.3% Triton X-100 solution for 15 min and treated with ammonium chloride 0.5 M for 15 min to reduce background. Cells were then blocked with PBS 2% BSA for 30 min, followed by 90 min of incubation with primary antibody against Ki-67 (1/100—M7240 Dako™). Cells were then washed with PBS and incubated with DAPI (1/20—Amersham PA53021) and a secondary antibody for 1 h (1/1500—Alexa568® anti-mouse). After washing with PBS, images were taken at a fluorescence microscope, and the Ki-67 positive ratio was assessed.
For SiR-Actin staining of the cells at the wound edge, cells were seeded on 96-well plates and grown until confluent. Cells were then wounded using a 96-pin tool, and wound closure was allowed for 16 h before cells were fixed and stained using SiR-Actin (Spirochrome, Switzerland) and Hoechst. Cells were imaged at 20× using a Leica SPE microscope (Leica, Jena, Germany).
For SiR-Actin staining of the cells at the wound edge, cells were seeded on 96-well plates and grown until confluent. Cells were then wounded using a 96-pin tool, and wound closure was allowed for 16 h before cells were fixed and stained using SiR-Actin (Spirochrome, Switzerland) and Hoechst. Cells were imaged at 20× using a Leica SPE microscope (Leica, Jena, Germany).
9
Visualizing Cells in FRESH Bioprinted Constructs
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To visualize cells adhering to FRESH bioprinted collagen constructs the cells were labelled overnight with SiR-actin (500 nM; Spirochrome, Switzerland), which permits fluorescence imaging of the actin cytoskeleton. Samples were washed twice in OptiMEM cell culture medium, which was then exchanged for OptiMEM containing the NucBlue nuclear stain. The cells were imaged as described above by confocal microscopy.
10
Caenorhabditis Genetics and Culture Protocols
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Heavy isotope labelled amino acids were purchased from Cambridge Isotope Laboratories Inc. (Andover, Massachusetts, USA), commercial sphingoid bases from Avanti Polar Lipids (Alabaster, Alabama, USA), except DL-C16-sphinganine from Matreya LLC (State College, Pennsylvania, USA), 12-methyl tridecanol from Endeavour Speciality Chemicals (Daventry, United Kingdom), SiR-actin from Spirochrome (Stein am Rhein, Switzerland), all other compounds and solvents at highest purity from Sigma-Aldrich (Buchs, Switzerland) or Acros Organics/Chemie Brunschwig (Basel, Switzerland), MH33 antibody was from DSHB (University of Iowa).
Wild-type N2 (var Bristol) and bacterial strains were received from the Caenorhabditis Genetics Centre (University of Michigan, Michigan, USA) VJ402 (fgEx11[ERM-1::GFP]) were a kind gift of Dr Verena Göbel. A complete list of bacteria, yeast strains and nematodes used in this study can be found inTable 1 . Unless stated otherwise bacteria were grown on LB medium, yeast on rich medium (YPUATD: 1% yeast extract, 2% bacto peptone, each 40 mg l–1 uracil, adenine, tryptophan, 2% glucose, 2% bacto agar) and worms on NGM medium seeded with OP50 bacteria.36 (link)
Wild-type N2 (var Bristol) and bacterial strains were received from the Caenorhabditis Genetics Centre (University of Michigan, Michigan, USA) VJ402 (fgEx11[ERM-1::GFP]) were a kind gift of Dr Verena Göbel. A complete list of bacteria, yeast strains and nematodes used in this study can be found in
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