Drosophila S2 cells were cultured in Schneider's Insect Medium (Gibco) supplemented with 10% FCS and antibiotics at 25°C (Schneider, 1972 (link)). pUAST vectors with actin5Ce-Gal4 drivers were cotransfected using Effectene (Qiagen) according to the manufacturer's instructions, and harvested 36-48 h after transfection. For immunofluorescence or time-lapse imaging, cells were replated on coverslips or glass-bottom dishes coated with Concanavalin A (Wako) and were allowed to spread for 1-2 h (Rogers et al., 2002 (link)). For drug treatments, cells were treated with 1 µM Latrunculin A (Wako) or 10 µM Colchicine (Wako) for 1 h before imaging. For immunofluorescence, cells were fixed in 4% paraformaldehyde in phosphate-buffered saline (PBS) for 20 min at room temperature, permeabilized with 0.1% Triton X-100 in PBS (PBS-T) for 15 min, and blocked with 5% skimmed milk in Tris-buffered saline (TBS). Primary and secondary antibodies were diluted in the blocking solution. After each antibody incubation, the coverslips were washed three times with PBS-T. The cells were mounted in Vectashield mounting medium (Vector Labs).
Latrunculin a
Manufactured by Fujifilm
Sourced in United Kingdom
Latrunculin A is a compound that inhibits the polymerization of actin filaments. It is commonly used as a research tool in cell biology laboratories to study the role of the actin cytoskeleton in various cellular processes.
Automatically generated - may contain errors
Lab products found in correlation
Dulbecco s modified eagle medium, by Fujifilm (2 mentions)
Penicillin streptomycin, by Fujifilm (2 mentions)
Eyfp actin vector, by Takara Bio (2 mentions)
Mclover2 c1 vector, by Addgene (2 mentions)
Myosin 2 atpase inhibitor blebbistatin, by Fujifilm (2 mentions)
Effectene, by Qiagen (1 mentions)
Schneider s insect medium, by Thermo Fisher Scientific (1 mentions)
Colchicine, by Fujifilm (1 mentions)
Concanavalin a (cona), by Fujifilm (1 mentions)
Vectashield mounting medium, by Vector Laboratories (1 mentions)
Dmem f12, by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
Trypsin edta, by Thermo Fisher Scientific (1 mentions)
Cholera toxin, by Merck Group (1 mentions)
Epidermal growth factor (egf), by Thermo Fisher Scientific (1 mentions)
Horse serum, by Thermo Fisher Scientific (1 mentions)
Insulin, by Merck Group (1 mentions)
Blebbistatin, by Merck Group (1 mentions)
Hydrocortizone, by Merck Group (1 mentions)
Lsm800 confocal laser microscope, by Zeiss (1 mentions)
6 protocols using latrunculin a
1
Culturing and Transfecting Drosophila S2 Cells
2
Visualizing Actin Dynamics in Smooth Muscle Cells
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Rat aortic smooth muscle cell lines (A7r5, ATCC) were cultured with low-glucose (1.0 g/L) Dulbecco’s Modified Eagle Medium (Wako) containing 10% (v/v) heat-inactivated fetal bovine serum (SAFC Biosciences) and 1% penicillin-streptomycin (Wako) in a humidified 5% CO2 incubator at 37°C. Expression plasmids encoding mClover2-tagged β-actin were constructed by inserting a human β-actin gene, which was digested with XhoI and BamHI restriction enzymes from the EYFP-actin vector (#6902–1, Clontech) into the mClover2-C1 vector (Addgene plasmid #54577, a gift from Michael Davidson) [16 (link)]. The plasmids were transfected to cells using Lipofectamin LTX with Plus Reagent (Thermo Fischer Science) according to the manufacturer’s instruction. Myosin II ATPase inhibitor (-)-blebbistatin (Wako) was used at 10 μM concentration, and actin polymerization inhibitor Latrunculin A (Wako) was used at 10 nM concentration. An equivalent amount of dimethyl sulfoxide (DMSO) was administered to cells as vehicle control.
3
Actin Polymerization and Depolymerization Kinetics
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Polymerisation of WT or K336I actin (10 µM) was induced by addition of concentrated F-buffer and the resultant increase in light scatter was monitored at 360 nm at 22 °C using a fluorescence spectrophotometer. The final concentration of each component was 100 mM KCl, 2 mM HEPES pH 7.4, 2.5 mM MgCl2, 0.5 mM EGTA, 0.5 mM ATP, and 0.5 mM DTT. Depolymerisation of WT (5 µM) or K336I (5 µM) actin filaments in buffer (100 mM KCl, 10 mM HEPES pH 7.4, 2.5 mM MgCl2, 0.5 mM EGTA, 0.1 mM ATP, and 1 mM DTT) was induced by addition of 36.5 µM Latrunculin A (Wako Chemicals). The critical concentration of actin was determined by polymerising various concentrations of actin (0.5, 1, 1.5, 3, 5, and 10 μM) in precipitation buffer (2 mM Tris-HCl, pH 8.0, 0.2 mM CaCl2, 50 mM KCl, 1 mM MgCl2, 1 mM DTT, and 0.2 mM ATP) for 20 minutes, followed by separation of the resultant F- and G-actin by centrifugation at 300,000 × g for 15 minutes at 4 °C. The concentration of each form was determined by quantitative densitometry of Coomassie-blue–stained SDS-PAGE gels.
4
MCF-10A Cell Culture Protocols
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MCF-10A human epithelial cells were cultured in growth medium composed of Dulbecco’s modified Eagle’s medium/Ham’s F-12 containing HEPES and L-glutamine (DMEM/F12, Invitrogen) supplemented with 5% horse serum (Invitrogen), 1% penicillin/streptomycin (Invitrogen), 10 µg/mL insulin (Sigma), 0.5 µg/mL hydrocortizone (Sigma), 20 ng/mL EGF (Peprotech) and 0.1 µg/mL cholera toxin (Sigma) and maintained under humidified conditions at 37 °C and 5% CO2. Cells were passaged regularly by dissociating confluent monolayers with 0.05% trypsin-EDTA (Invitrogen) and suspending cells in DMEM/F12 supplemented with 20% horse serum and 1% penicillin/streptomycin. Cells were passaged at 1:4 in growth medium.
Other non-cancerous cells tested were RWPE1 (epithelial) and L929 (fibroblast); cancer cells tested were MCF-7 (epithelial), RWPE2 (prostate), PC3 (prostate), H292 (lung) and THP-1(leukemia)-derived macrophages. As for the genetically modified MCF-10A variants, MCF-10A APC−/− were purchased from Sigma Aldrich, while the MCF-10A RAS was transformed via introduction of the HrasV12 oncogene39 (link). They were all cultured in accordance with the ATCC recommended media and passage protocols. Also, blebbistatin (Sigma) or latrunculin A (Wako) dissolved in dimethylsulfoxide (DMSO) was used at 5 μM in growth media for the myosin or the actin polymerization inhibition experiments, respectively.
Other non-cancerous cells tested were RWPE1 (epithelial) and L929 (fibroblast); cancer cells tested were MCF-7 (epithelial), RWPE2 (prostate), PC3 (prostate), H292 (lung) and THP-1(leukemia)-derived macrophages. As for the genetically modified MCF-10A variants, MCF-10A APC−/− were purchased from Sigma Aldrich, while the MCF-10A RAS was transformed via introduction of the HrasV12 oncogene39 (link). They were all cultured in accordance with the ATCC recommended media and passage protocols. Also, blebbistatin (Sigma) or latrunculin A (Wako) dissolved in dimethylsulfoxide (DMSO) was used at 5 μM in growth media for the myosin or the actin polymerization inhibition experiments, respectively.
5
Investigating Micropinocytosis Inhibition on siRNA Uptake
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The effects of inhibition of micropinocytosis using endocytosis inhibitors on anti-CD63 mAb-conjugated siRNA complex uptake into MM cells were investigated. OPM-2-luc cells were seeded in a 96-well plate at 1.5 × 104/well. After 24 h incubation, cells were transferred to glass-bottomed dishes and then treated with endocytosis inhibitors, Rottlerin [27 (link)] (Abcam, Cambridge, UK) and Latrunculin A [28 (link),29 (link)] (FUJIFILM Wako Pure Chemical Corporation), at 2 μM for 45 min. Then, cells were treated with anti-CD63 mAb-conjugated luc2 siRNA labeled with FITC. Twenty-four hours after ADC treatment, OPM-2-luc cells were stained with Hoechst 33342 (5 μM) for 10 min at room temperature in the dark. Fluorescence images were acquired on an LSM800 laser confocal microscope (Carl Zeiss).
6
Rat Aortic Smooth Muscle Cell Imaging
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Rat aortic smooth muscle cell lines (A75r, ATCC) were cultured with low-glucose (1.0 g/L)
Dulbecco's Modified Eagle Medium (Wako) containing 10% (v/v) heat-inactivated fetal bovine serum (SAFC Biosciences) and 1% penicillin-streptomycin (Wako) in a humidified 5% CO2 incubator at 37℃. Expression plasmids encoding mClover2-tagged β-actin were constructed by inserting a human β-actin gene, which was digested with XhoI and BamHI restriction enzymes from the EYFP-actin vector (#6902-1, Clontech) into the mClover2-C1 vector (Addgene plasmid #54577, a gift from Michael Davidson). The plasmids were transfected to cells using Lipofectamin LTX with Plus Reagent (Thermo Fischer Science) according to the manufacturer's instruction. Myosin II ATPase inhibitor (-)-blebbistatin (Wako) was used at 10 μM concentration, and actin polymerization inhibitor Latrunculin A (Wako) was used at 10 nM concentration. An equivalent amount of dimethyl sulfoxide (DMSO) was administered to cells as vehicle control.
Dulbecco's Modified Eagle Medium (Wako) containing 10% (v/v) heat-inactivated fetal bovine serum (SAFC Biosciences) and 1% penicillin-streptomycin (Wako) in a humidified 5% CO2 incubator at 37℃. Expression plasmids encoding mClover2-tagged β-actin were constructed by inserting a human β-actin gene, which was digested with XhoI and BamHI restriction enzymes from the EYFP-actin vector (#6902-1, Clontech) into the mClover2-C1 vector (Addgene plasmid #54577, a gift from Michael Davidson). The plasmids were transfected to cells using Lipofectamin LTX with Plus Reagent (Thermo Fischer Science) according to the manufacturer's instruction. Myosin II ATPase inhibitor (-)-blebbistatin (Wako) was used at 10 μM concentration, and actin polymerization inhibitor Latrunculin A (Wako) was used at 10 nM concentration. An equivalent amount of dimethyl sulfoxide (DMSO) was administered to cells as vehicle control.
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