10,000 (A375, Cal51) or 20,000 (MDA-MB-231) cells of interest were plated in 250 μl of media in a flat-bottomed 24-well plate and allowed to attach for 24 hr. Then the media was replaced, and OTS167 or Cytochalasin B (Cayman Chemical Company, Ann Arbor, MI; Cat. No. 11328) were added to control wells. Following an additional 24 hr period of growth, cells were stained with 2.5 μg/ml of Hoechst dye (Thermo Fisher, Cat. No. H3569) for 30 min and imaged using appropriate filters. DNA staining experiments were performed twice.
Cytochalasin b
Manufactured by Cayman Chemical
Sourced in France
Cytochalasin B is a biochemical product offered by Cayman Chemical. It is a fungal metabolite that functions as an inhibitor of actin polymerization. This product is intended for research use only.
Automatically generated - may contain errors
Lab products found in correlation
M mlv reverse transcriptase, by Thermo Fisher Scientific (2 mentions)
Trizol reagent, by Thermo Fisher Scientific (2 mentions)
Dulbecco phosphate buffered saline (dpbs), by Thermo Fisher Scientific (2 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (2 mentions)
Hoechst dye, by Thermo Fisher Scientific (1 mentions)
Net549a001mc, by PerkinElmer (1 mentions)
Cyclophosphamide, by Merck Group (1 mentions)
Mitomycin c, by Cayman Chemical (1 mentions)
Dpx mounting medium, by Merck Group (1 mentions)
Methanol, by Merck Group (1 mentions)
Glacial acetic acid, by Merck Group (1 mentions)
Mushroom and yeast beta glucan assay kit, by Megazyme (1 mentions)
Etoposide, by Cayman Chemical (1 mentions)
Nu7441, by Cayman Chemical (1 mentions)
Cisplatin, by Merck Group (1 mentions)
Calf bovine serum, by Thermo Fisher Scientific (1 mentions)
Human insulin, by Merck Group (1 mentions)
Emetine, by Merck Group (1 mentions)
Bodipy 493 503, by Cayman Chemical (1 mentions)
Deoxy d glucose 2 1 2 3h n, by American Radiolabeled Chemicals (1 mentions)
5 protocols using cytochalasin b
1
Cell Proliferation Assay with Cytoskeletal Inhibitors
2
Measuring Glucose Uptake in BMDMs
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BMDMs were plated at a density of 1 million cells per well in 12-well plates and stimulated as indicated. Cells were then washed with Krebs-Ringer bicarbonate HEPES (KRBH) buffer and then given 400 μL with KRBH buffer loaded with 0.8 μCi/well [3H]−2-deoxyglucose (PerkinElmer, NET549A001MC) and 0.5 mM unlabeled 2-deoxyglucose and incubated at 37°C for 30 min. 10 μL of 1.5 mM Cytochalasin B (Cayman Chemical, 11328) was then added to stop glucose uptake. 400 μL of lysate was used to measure levels of [3H]−2-deoxyglucose by a scintillation counter, and the remaining lysate was used to measure total protein content for normalization.
3
Isolation and Analysis of Beta-Glucan
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Ham’s F-10 was purchased from Biosera (Nuaille, France), mitomycin C and cytochalasin B were purchased from Cayman chemical company (Ann Arbor, MI, United States), Mushroom and Yeast Beta-Glucan assay kit (K-YBGL) was purchased from Megazyme (Bray, Ireland), Luna Universal PCR mix was purchased from New England Biolabs Inc. (Ipswich, MA, United States), Giemsa’s azur eosin methylene blue solution was purchased from Panreac Applichem (Castella del Valles, Spain), cyclophosphamide, DPX mounting medium, methanol, glacial acetic acid and formalin solution (neutral buffered, 10%) were purchased from Merck (Darmstadt, Germany), Fetal Bovine Serum (FBS), L -glutamine, M-MLV reverse transcriptase, Phytohemagglutinin M (PHA-M), and Trizol reagent were purchased from Thermo Fisher Scientific (Paisley, United Kingdom).
4
Preparation and Storage of Anticancer Agents
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Cisplatin (Sigma-Aldrich 479306) was dissolved in sterile 0.9% NaCl to a 3-mM concentration and stored at −80 °C until use, avoiding thawing/freezing cycles. Etoposide (Cayman Chemical Company, Ann Arbor, MI, USA; n. 12092) was dissolved in dimethyl sulfoxide (DMSO) to 50 mM and stored in aliquots at −20 °C. Bleomycin sulfate (Molekula Group, Darlington, UK; n. 25097489) was dissolved in sterile 0.9% NaCl to a 0.5-mM concentration and stored in aliquots at −80 °C. NU7441 (Cayman chemical company n. 14881) was dissolved in DMSO to a 5-mM concentration and stored in aliquots at −20 °C. The PARPi AZD2281 was provided as powder by the Memorial Sloan-Kettering Cancer Center and dissolved in DMSO to a concentration range of 10−8 to 10−4 M and stored in aliquots at −20 °C. Cytochalasin B (Cayman chemical company; 11328) was dissolved in ethanol to a 50-mM concentration and stored in aliquots at −20 °C.
5
Insulin Signaling Pathway Visualization
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Human insulin and emetine were purchased from Sigma-Aldrich (St. Louis, MO); cytochalasin B, BODIPY 493/503, actinomycin D -from Cayman Chemical (Ann Arbor, MI) and fetal bovine serum (FBS) -from Atlanta Biologicals (Lawrenceville, GA). Calf bovine serum (CBS), ProLong® Gold antifade reagent with DAPI, Halt™ Protease and Phosphatase Inhibitor Cocktail, DMEM and DPBS -from Thermo Fisher Scientific (Waltham, MA). Deoxy-D-glucose, 2-[1,2-3H(N)] was purchased from American Radiolabeled Chemicals (St. Louis, MO). Mouse monoclonal antibodies against actin and myc tag, rabbit monoclonal antibody against phospho-TBC1D4 (Thr642), rabbit polyclonal antibodies against the myc tag, phospho-Akt (Ser473 or Thr308), TBC1D4, HRP-conjugated anti-mouse and anti-rabbit IgG were purchased from Cell Signaling Technology (Danvers, MA). Mouse monoclonal anti-syntaxin 6 antibody was purchased from BD Biosciences (Franklin Lakes, NJ). Rabbit polyclonal antibody against TUG were described previously 11, 12 (link) . Alexa Fluor 594-conjugated goat anti-mouse antibody, Alexa Fluor 488conjugated goat anti-rabbit antibody and Alexa Fluor 647-conjugated mouse transferrin were purchased from Jackson ImmunoResearch (West Grove, PA).
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