For formin drug inhibition studies, cells were incubated with 20 or 40 μM SMIFH2 (4401, TOCRIS, UK) in serum containing DMEM for 1–2 h at 5% CO2, 37 °C. In in vitro experiments, SMIFH2 (340316-62-3, Sigma-Aldrich) was used at a concentration of 100 μM (see below). For myosin II inhibition studies, 30 μM or 50 μM Rho-kinase (ROCK) inhibitor Y-27632 (Y0503, Sigma-Aldrich), or 20 μM S-nitro-blebbistatin (13013-10, Cayman Chemicals) was added for 10–20 min before the experiments or directly during observation. All inhibitors remained in the medium during the entire observation period.
S nitro blebbistatin
Manufactured by Cayman Chemical
(S)-nitro-blebbistatin is a selective myosin II inhibitor. It is used as a research tool to study the role of myosin II in various cellular processes.
Automatically generated - may contain errors
Lab products found in correlation
Smifh2, by Merck Group (4 mentions)
Y 27632, by Merck Group (2 mentions)
Nsc23766, by Merck Group (2 mentions)
Devd fmk conjugated to sulfo rhodamine, by Abcam (2 mentions)
Sir actin, by Cytoskeleton (2 mentions)
Hoechst 33342, by Merck Group (2 mentions)
Latrunculin b, by Merck Group (2 mentions)
Smifh2, by Bio-Techne (1 mentions)
Ouabain, by Bio-Techne (1 mentions)
Plan apochromatic 100 silicone, by Nikon (1 mentions)
Calyculin a, by Cayman Chemical (1 mentions)
Cytochalasin d, by Bio-Techne (1 mentions)
Y 27632, by Hello Bio (1 mentions)
Blebbistatin, by Cayman Chemical (1 mentions)
Hydrochloric acid (hcl), by Thermo Fisher Scientific (1 mentions)
Sodium hydroxide (naoh), by Thermo Fisher Scientific (1 mentions)
Cytochalasin d, by Merck Group (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
Nucleofector device, by Lonza (1 mentions)
Growth supplement, by Cell Applications (1 mentions)
9 protocols using s nitro blebbistatin
1
Formin and Myosin II Inhibition Protocol
2
Actin and Ezrin Inhibitor Treatments
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Cells were treated with the actin inhibitor cytochalasin-D (5 µM; Tocris), the ezrin inhibitor NSC668394 (50 µM; Calbiochem), Y27632 (25 µM; Hello Bio), calyculin A (10 nM; Cayman Chemical), Ouabain (30 nM; Tocris), and s-nitro-Blebbistatin 25 µM; Cayman Chemical). For cytochalasin-D and calyculin A treatment, an 18-slice Z-stack (140 nm step size) was acquired every minute for 5 min using a Plan Apochromatic 100× silicone oil immersion objective (Nikon; NA 1.35) as described above. After 5 min, a solution of cytochalasin-D dissolved in imaging media was injected into the well and the cells were imaged for another 15 min posttreatment. For NSC668394, Y27632, Ouabain, and S-nitro- Blebbistatin, cells were treated immediately before imaging and an 18-slice Z-stack (192.5 nm step size) was acquired every hour for 17 h using a Plan Apochromatic 40× air objective (Nikon; NA 0.95) and a 2× zoom lens (80× total magnification). Each drug treatment experiment was repeated in three separate wells, and multiple fields of view were collected per well.
3
Cardiomyocyte Culture Optimization
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M1018 was made according to the manufacturer’s directions, using cell culture–grade NaHCO3 and penicillin-streptomycin-glutamate (Mediatech). Medium was further supplemented, as indicated, with 10 mM BDM, 0.5 µM cytochalasin D, 10 mM HEPES, 0.2% BSA, 10% FBS, 5 mM taurine, and/or 1× insulin-transferrin-selenium (ITS; Thermo Fisher Scientific). pH was adjusted to 7.35 or 7.8 using NaOH (Thermo Fisher Scientific) and HCl (Thermo Fisher Scientific). All media were 0.22-µm sterile filtered before use. (-)-Blebbistatin (Cayman) or (S)-nitro-blebbistatin (Cayman) was dissolved to a concentration of 25 mM in filtered, cell culture–grade DMSO before being further diluted, as indicated, into the medium.
4
Activation and Inhibition of T-cell Signaling
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Cytochalasin-D and latrunculin-B were obtained from EMD Millipore, (S)-nitro-Blebbistatin was purchased from Cayman Chemical, CK666 was from Calbiochem, and Y27632 and SMIFH2 were from Sigma-Aldrich. Flow cytometry antibodies: rat anti-CD4 APC/APC-Cy7 (clone RM4-5), rat anti-CD8a APC/PE-Cy7 (clone 53–6.7), Armenian hamster anti-CD69 APC (clone H1.2F3) and rat anti-CD25 PE (clone PC61) were all purchased from BioLegend. Surface coating antibodies: Armenian hamster anti-CD3ε (clone 2C11) and Armenian hamster anti-CD28 (Clone PV1) were from BioXCell. Biotinylated Armenian hamster anti-CD3ε (clone 2C11) was from Invitrogen, and biotinylated mouse anti-TCRVβ5.1/5.2 (clone MR9-4) was from BD Bioscience. Dendritic cell staining: anti-CD86 CF555 was made by conjugating purified rat anti-CD86 (BioXCell) with CF555 conjugated dye from Biotium as per the manufacturer’s protocol.
5
Live-Cell Imaging of Cytoskeletal Dynamics
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Drugs were incubated 2 h prior to imaging unless stated otherwise. The drug concentrations are as follow: (S)-nitro-blebbistatin (Cayman Chemical) 50 μM, NSC-23766 (Sigma–Aldrich) 200 µM, SMIFH2 (Sigma) 50 μM dissolved in DMSO. Caspase-3 indicator DEVD-FMK conjugated to Sulfo-rhodamine (Abcam) was used at 1:1000 dilution and incubated with the cells 30 min before experiment. To visualize F-actin in certain experiments, siR-actin (Cytoskeleton) was added at 100 nM concentration to the medium 12 h before imaging. Hoechst 33342 at 1 µg/mL (Sigma) was added into culture media 1 h and washed before imaging for nucleus live detection.
6
Nitro-Blebbistatin Imaging Preparation
7
HUVEC Cytoskeleton Dynamics Imaging
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Human umbilical vein endothelial cells (HUVECs) were cultured in EC basal medium (EBM) supplemented with growth supplements (Cell Applications) and penicillin–streptomycin (Fisher) and maintained at 37°C in 5% CO2. All transfections were performed in Mirus Nucleofection buffer using a Lonza Nucleofector Device, setting A-034. ECs were cultured at 75,000–100,000 cells/coverslip and plated on 10 µg/ml fibronectin-coated 35 mm glass bottom dishes (Cat. #: P35G-1.0-20-C.s). Transfections were imaged 3 h after experimental cDNA transfections unless indicated otherwise (for MT group prep, see below). For nocodazole experiments, cells were treated with 10 µM nocodazole for 20 min and washed out with EBM before imaging. Cells treated with s-nitro blebbistatin (20 µM; Cayman Chemicals) in dimethyl sulfoxide (DMSO; 0.001%) were monitored every 20 min for 60 min before being imaged at 60 min. Blebbistatin washout experiments were done with >6 EBM changes. Latrunculin experiments were done at 1.25 µM resuspended in DMSO; 0.001%. Cells were treated on the microscope stage and imaged immediately after.
8
Multimodal Imaging of Actin Dynamics
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Drugs were incubated 2 hours prior to imaging unless stated otherwise. The drug concentrations are as follow: (S)-nitro-blebbistatin (Cayman Chemical) 50 μM, NSC-23766 (Sigma-Aldrich) 200 µM, SMIFH2 (Sigma) 50 μM dissolved in DMSO. Caspase-3 indicator DEVD-FMK conjugated to Sulfo-rhodamine (Abcam) was used at 1:1000 dilution and incubated with the cells 30 min before experiment. To visualize F-actin in certain experiments, siR-actin (Cytoskeleton) was added at 100 nM concentration to the medium 12 hours before imaging. Hoechst 33342 at 1µg/mL (Sigma) was added into culture media 1 hour and washed before imaging for nucleus live detection.
9
Immune Cell Activation Pathway Profiling
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