Cardiac myocytes were plated onto laminin-coated 10 cm culture dishes (400,000–500,000 rod-shaped cardiomyocytes per dish) in plating media. After incubating the cells under 21% O2 and 2% CO2 condition for more than 1 h until rod-shaped cells were attached, the plating medium was changed to culture medium [minimum essential medium with Hank’s balanced salt solution, supplemented with (S)-(−)-blebbistatin (25 μM, Toronto Research Chemicals), insulin-transferrin-sodium selenite media supplement (10 μg/mL, 5.5 μg/mL, and 5 ng/mL, respectively), fatty-acid free bovine serum albumin (1 mg/mL, Millipore), penicillin-streptomycin (100 units/mL), and glutamine (2 mM)], and the cells were cultured for 1–2 h or overnight under 21% O2 and 2% CO2 condition. For hypoxia-reoxygenation treatment, cardiomyocytes were treated with 10 mM 2-deoxyglucose followed by incubating under 1% O2 and 2% CO2 condition for 6 h using the multi-gas incubator. After the hypoxia period, culture media was changed to the fresh one without 2-deoxyglucose, and incubate cells under 1% O2 and 2% CO2 condition for reoxygenation. After the reoxygenation period, cells were collected for further analyses.
S blebbistatin
Manufactured by LGC
Sourced in Japan
(S)-(-)-blebbistatin is a small molecule that acts as a selective inhibitor of myosin II ATPase activity. It is a chemical tool commonly used in biological research to study the role of myosin II in various cellular processes.
Automatically generated - may contain errors
Lab products found in correlation
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
Insulin transferrin selenium supplement, by Thermo Fisher Scientific (1 mentions)
Cytochalasin d, by Merck Group (1 mentions)
Mouse anti gapdh ca92590 ab, by Merck Group (1 mentions)
Withaferin a, by Santa Cruz Biotechnology (1 mentions)
Jte 013, by Merck Group (1 mentions)
Pf 4708671, by Merck Group (1 mentions)
U0126, by Promega (1 mentions)
C3755, by Merck Group (1 mentions)
4 protocols using s blebbistatin
1
Cardiac Myocyte Hypoxia-Reoxygenation Assay
2
Perfusion and EDTA Protocol with Blebbistatin
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Perfusion and EDTA buffers were prepared as previously described2 (link), except that they contained 15 µmol L−1 (S)-(−)-blebbistatin (Toronto Research Chemicals, Toronto, ON). EDTA, taurine, and HEPES were supplied by BioShop Inc. (Burlington, ON). M199 (Wisent Inc., Saint-Jean-Baptiste, QC) pH 7.6 was supplemented with added 100X CD lipid, 100X insulin-transferrin-selenium supplement (Life Technologies), and 100X penicillin/streptomycin to 1X each. All other reagents unless mentioned were supplied by Sigma-Aldrich (St. Louis, MI).
3
Immunoblotting and Immunofluorescence Assays
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Mouse anti-Myc (sc-40) and rabbit anti-YAP (sc-10547) antibodies (Abs) were purchased from Santa Cruz, mouse anti-GAPDH (CA92590) Ab was from Merck Millipore, and mouse anti-filamin (F6682) Ab was from Sigma-Aldrich. For immunoblotting, Abs were used at 1:3000 (anti-Myc), 1:1000 (anti-YAP) and 1:5000 (anti-GAPDH) dilution, respectively. For immunofluorescence, anti-filamin Ab was used at 1:100 dilution. The chemical inhibitors cytochalasin D (0.4 μM), JTE-013 (10 μM) and PF-4708671 (10 μM) were purchased from Sigma-Aldrich; bisindolylmaleimide I (10 μM), LY294002 (10 μM) and rapamycin (0.1 μM) from Calbiochem; withaferin A (2.5 μM) from Santa Cruz; U0126 (10 μM) from Promega; and (S)-(-)-blebbistatin (30 μM) from Toronto Research Chemicals. Inhibitors and Dox were purchased from Apollo Scientific and were added simultaneously to cultures.
4
Relaxing and Activating Solutions for Muscle Fiber Experiments
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The composition of the solutions was as described [24 (link)]. The relaxing solution contained 80 mM K-propionate, 20 mM imidazole, 10 mM EGTA, 4 mM ATP, 5 mM MgCl2, 20 mM phosphocreatine and 300 U/ml creatine phosphokinase (C3755, Sigma-Aldrich, USA) (pH=7.2). In the pre-activating solution, the concentration of EGTA was reduced to 0.2 mM. The activating solution contained 10.1 mM CaCl2 in addition to the components of the relaxing solution. BTS was obtained from Sigma Rare Chemicals (USA) or Tokyo Chemical Industry Co. (Japan), and (S)-(−)-blebbistatin was obtained from Toronto Research Chemicals (Canada) or Calbiochem (USA). BDM was obtained from Sigma-Aldrich. When necessary, these chemicals were added to the three solutions described above.
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