Murine EL4 (EC85023105) T cells were purchased from the European Collection of Cell Cultures. EL4 T cell is a murine thymoma cell line that retains many T-cell properties and is widely used for studying T-cell signaling and transcriptional regulation in T cells (45 (link), 46 (link)). The cells (1 mL at 106 cells/well) were cultured in RPMI 1640 medium (Wako Pure Chemicals Industries Ltd., Osaka, Japan) containing 10% FCS (Nichirei Bioscience Inc., Tokyo, Japan), and penicillin/streptomycin (Wako Pure Chemicals Industries Ltd.), either in the presence or absence of vinblastine, nocodazole, and cytochalasin D (Wako Pure Chemicals Industries Ltd.). The viability of EL4 T cells was calculated before and after the drug treatments using a Countess® Automated Cell Counter (Life Technologies, Gaithersburg, MD, USA), according to the manufacturer’s instructions, as previously described (47 (link)). Data were collected from 3 independent experiments. Student t-tests were utilized to detect significant differences. Data are presented as mean ± SE, and a p value < 0.05 was considered statistically significant.
Cytochalasin d
Manufactured by Fujifilm
Sourced in Japan
Cytochalasin D is a small molecule compound used in cell biology research. It functions as an inhibitor of actin polymerization, disrupting the dynamic cytoskeleton of cells.
Automatically generated - may contain errors
Lab products found in correlation
Rottlerin, by Nacalai Tesque (11 mentions)
Dynasore, by Nacalai Tesque (10 mentions)
Isoflurane, by Fujifilm (9 mentions)
Nystatin, by Merck Group (7 mentions)
Nocodazole, by Fujifilm (5 mentions)
Propyl p hydroxybenzoate, by Fujifilm (4 mentions)
L menthol, by Fujifilm (4 mentions)
Pentobarbital, by Tokyo Chemical Industry (3 mentions)
Methyl p hydroxybenzoate, by Fujifilm (3 mentions)
Protein assay kit, by Bio-Rad (3 mentions)
Methylcellulose mc, by Shin-Etsu Chemical (3 mentions)
Penicillin, by Thermo Fisher Scientific (3 mentions)
Carbopol 934, by Serva Electrophoresis (3 mentions)
Streptomycin, by Thermo Fisher Scientific (2 mentions)
Benzalkonium chloride bac, by Kanto Chemical (2 mentions)
Indomethacin powder, by Fujifilm (2 mentions)
Mannitol d mannitol, by Fujifilm (2 mentions)
4 2 hydroxyethyl 1 piperazineethanesulfonic acid hepes buffer, by Fujifilm (2 mentions)
Blebbistatin, by Fujifilm (2 mentions)
Nystatin, by Serva Electrophoresis (2 mentions)
28 protocols using cytochalasin d
1
Murine EL4 T Cell Culture Protocols
2
Formulation and Characterization of Nanoparticles
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FAM powder, polyvinylpyrrolidone (PVP), gum arabic, isoflurane, D-mannitol, methyl p-hydroxybenzoate, and cytochalasin D were purchased from Wako Pure Chemical Industries Ltd. (Osaka, Japan). The Bio-Rad Protein Assay Kit and 2-hydroxypropyl-β-cyclodextrin (HPβCD) were obtained from Bio-Rad (CA, United States) and Nihon Shokuhin Kako Co., Ltd. (Tokyo, Japan), respectively. Nystatin was purchased from Sigma–Aldrich (St. Louis, MO, United States). Pentobarbital and 1-heptanesulfonic acid sodium salt were obtained from Tokyo Chemical Industry Co. Ltd. (Tokyo, Japan). Methylcellulose (MC) was purchased from Shin-Etsu Chemical Co. Ltd. (Tokyo, Japan). Rottlerin and dynasore were obtained from Nacalai Tesque (Kyoto, Japan). All chemicals used were of the highest commercially available purity.
3
Plaque Assay for Oncolytic Virus
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Cells were plated at 2 × 105/well in 6-well plates. After 36 h, cells were infected with OVV-LG at an MOI of 0.001. After a 1-h incubation, E-MEM containing 0.8% methyl cellulose supplemented with 5% FBS and reagents was added to each well, and plaque sizes were calculated with a BZ-X700 microscope. Reagents used for plaque assays were as follows: nocodazole (Wako, Osaka, Japan), colchicine (Wako), cytochalasin D (Wako), Rhosin (Merck, Boston, MA, USA), ML-141 (Sigma, St. Louis, MO, USA), and NSC23766 (Abcam, Cambridge, UK).
4
Fc Fragment Modulation Techniques
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Human IgG F(ab’)2 fragments (Gamma Venin P) were purchased from Sanofi, Paris, France. N-acetyl cysteine (NAC) (Sigma, St Louis, MO), pyrrolidine dithiocarbamate (PDTC) (Abcam), TPCA-1 (Abcam), methyl-β-cyclodextrin (Sigma) and cytochalasin D (Wako, Osaka, Japan) were also purchased. Human BD Fc Block was purchased from BD Biosciences, Franklin Lakes, NJ.
5
Activators and Inhibitors of TRPC Channels
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The following TRPC3/C6/C7 activators were employed in the current study: L687 (Supplementary Figure S1 , WO/2022/118966) (29 ), Cannabidiol (CBD) (#Axon1234; Axon Medchem, VA, USA), and GSK1702934A (#6508; Tocris Bioscience, Bristol, UK). SKF96365 (#1147/10; R&D Systems, MN, USA) was used as the TRPC channel inhibitor. BAPTA-AM (#B035; DOJINDO, Kumamoto, Japan) was used as the Ca2+ chelator. EIPA (#14406; Funakoshi Co., Tokyo, Japan) and Cytochalasin D (#034-25881; FUJIFILM Wako Pure Chemical Co., Osaka, Japan) were used as the macropinocytosis inhibitors. Nifedipine (#141-05783; FUJIFILM Wako Pure Chemical Co., Osaka, Japan) was used as the L-type calcium channel blocker. UNC7938 (#AOB13597; AOBIOUS Inc., MA, USA) was used as the destabilizer of the endosomal membrane. All reagents were dissolved in anhydrous DMSO (#D12345; Thermo Fisher Scientific, MA, USA) and added to the medium at a final concentration of 0.3–1% (v/v).
6
Synthesis and Characterization of Polymer Nanoparticles
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The
NIPAAm provided by
KJ Chemicals Co. (Tokyo, Japan) was purified by recrystallization
from n-hexane and dried at room 25 °C in vacuo.
The N,N′-dimethylaminopropylacrylamide
(DMAPAAm) was purchased from KJ Chemicals Co. (Tokyo, Japan), and
2,2′-azobisisobutyronitrile (AIBN), 3-mercaptopropionic acid
(MPA), sucrose, cytochalasin D, nocodazole, and DOPE were purchased
from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). N-Hydroxysuccinimide (NHS) and N,N′-dicyclohexylcarbodiimide (DCC) were purchased from Kanto
Chemical Co. (Tokyo, Japan). 5(6)-CF and chlorpromazine were purchased
from Sigma-Aldrich Corp. (St. Louis, MO). Filipin III was purchased
from Cayman Chemical (Ann Arbor, MI). Dulbecco’s modified Eagle’s
medium was purchased from Thermo Fisher Scientific (Waltham, CA),
and the fetal bovine serum (FBS) was purchased from Biosera (Sussex,
U.K.). The rhodamine B 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine
triethylammonium salt (rhodamine–DHPE) was purchased from Life
Technologies Co. (Carlsbad, CA), and DOTAP and N-[methoxy
(PEG) 2000]–distearoylphosphatidylethanolamine (PEG–DSPE)
were purchased from Avanti Polar Lipids Inc. (Alabaster, AL). All
other reagents and solvents were of analytical grade.
NIPAAm provided by
KJ Chemicals Co. (Tokyo, Japan) was purified by recrystallization
from n-hexane and dried at room 25 °C in vacuo.
The N,N′-dimethylaminopropylacrylamide
(DMAPAAm) was purchased from KJ Chemicals Co. (Tokyo, Japan), and
2,2′-azobisisobutyronitrile (AIBN), 3-mercaptopropionic acid
(MPA), sucrose, cytochalasin D, nocodazole, and DOPE were purchased
from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). N-Hydroxysuccinimide (NHS) and N,N′-dicyclohexylcarbodiimide (DCC) were purchased from Kanto
Chemical Co. (Tokyo, Japan). 5(6)-CF and chlorpromazine were purchased
from Sigma-Aldrich Corp. (St. Louis, MO). Filipin III was purchased
from Cayman Chemical (Ann Arbor, MI). Dulbecco’s modified Eagle’s
medium was purchased from Thermo Fisher Scientific (Waltham, CA),
and the fetal bovine serum (FBS) was purchased from Biosera (Sussex,
U.K.). The rhodamine B 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine
triethylammonium salt (rhodamine–DHPE) was purchased from Life
Technologies Co. (Carlsbad, CA), and DOTAP and N-[methoxy
(PEG) 2000]–distearoylphosphatidylethanolamine (PEG–DSPE)
were purchased from Avanti Polar Lipids Inc. (Alabaster, AL). All
other reagents and solvents were of analytical grade.
7
Nanomaterial-Based Drug Delivery Protocol
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Indomethacin powder (particle size, 5.01±0.59 µm), cytochalasin D, isoflurane, mannitol (D-mannitol) and propyl p-hydroxybenzoate were purchased from Wako Pure Chemical Industries, Ltd (Osaka, Japan). 2-Hydroxypropyl-β-cyclodextrin (HPβCD) was obtained from Nihon Shokuhin Kako Co., Ltd (Tokyo, Japan), and nystatin and rat tail collagen type 1, was provided by Sigma-Aldrich Japan (Tokyo, Japan). Methylcellulose (MC, type SM-4) with an average viscosity of ~4 Pa·s at 20°C was obtained from Shin-Etsu Chemical Co., Ltd (Tokyo, Japan). Dynasore and rottlerin were purchased from Nacalai Tesque (Kyoto, Japan), and benzalkonium chloride (BAC) was provided by Kanto Chemical Co., Inc. (Tokyo, Japan). Dulbecco’s Modified Eagle’s Medium/Ham’s F12, heat-inactivated fetal bovine serum, streptomycin and penicillin were obtained from Gibco (Thermo Fisher Scientific, Waltham, MA, USA). TetraColor One was purchased from Seikagaku Co. (Tokyo, Japan). All other chemicals used were of the highest purity commercially available.
8
Topical Delivery of Terbinafine Hydrochloride
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Pentobarbital was purchased from Sumitomo Dainippon Pharma Co., Ltd. (Toyo, Japan). TUL, cytochalasin D, methyl p-hydroxybenzoate, polyethylene glycol (PEG) 4000, cetyl alcohol, l-menthol, mineral oil, white wax, sodium tetraborate, propylene glycol, beeswax, and sodium dodecyl sulfate were provided by Wako Pure Chemical Industries, Ltd. (Osaka, Japan). Commercially available 0.5 mg TUL tape (CA-TUL, Hokunalin® Tapes 0.5 mg) was obtained from Mylan EPD G.K (Tokyo, Japan). Rottlerin and dynasore were purchased from Nacalai Tesque (Kyoto, Japan), and methylcellulose (MC, SM-4) was obtained from Shin-Etsu Chemical Co., Ltd. (Tokyo, Japan). 2-hydroxypropyl-β-cyclodextrin (HPβCD) was supplied by Nihon Shokuhin Kako Co., Ltd. (Tokyo, Japan), and PEG 400 was provided by Maruishi Pharmaceutical Co., Ltd. (Osaka, Japan). Nystatin and 450 nm pore size MFTM membrane filters were purchased from Sigma-Aldrich Japan (Tokyo, Japan) and Merck Millipore (Tokyo, Japan), respectively. All other chemicals used were of the highest purity commercially available.
9
Comprehensive Reagents for Cell Culture
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Dulbecco’s Modified Eagle’s Medium (high-glucose, DMEM), non-essential amino acids solution, rottlerin, and dynasore were purchased from Nacalai Tesque (Kyoto, Japan), and heat-inactivated fetal bovine serum, penicillin, and streptomycin were obtained from Grand Island Biological Company (GIBCO, Tokyo, Japan). l -glutamine and nystatin were purchased from Sigma-Aldrich Japan (Tokyo, Japan). Indomethacin powder (particle size, 16.3 ± 12.9 μm), 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer, propyl p-hydroxybenzoate, isoflurane, and cytochalasin D were provided by Wako Pure Chemical Industries, Ltd. (Osaka, Japan). HPβCD and MC (type SM-4) were supplied by Nihon Shokuhin Kako Co., Ltd. (Tokyo, Japan) and Shin-Etsu Chemical Co., Ltd. (Tokyo, Japan), respectively. Pentobarbital was obtained from Tokyo Chemical Industry Co., Ltd (Tokyo, Japan), and methanol and acetonitrile were provided by Kanto Chemical Co., Inc. (Tokyo, Japan). The Bio-Rad Protein Assay Kit was purchased from Bio-Rad Laboratories, Inc. (Bio-Rad, CA, U.S.A.). All other chemicals were of the highest purity which was commercially available.
10
Cell Spreading and Differentiation Protocols
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For cell spreading studies, the following concentrations were used: 50 µM blebbistatin, 30 µM Y-27632, 1 µM Cytochalasin D (all from Wako, Japan) and 2 mM GRGDS (Peptide institute, Japan). Each of these reagents was added 2 h prior to analysis. For lipid raft and pFAK studies, 25 µM methyl-β-cyclodextrin (MβCD, Santa Cruz Biotechnology) was added when seeding the cells. MβCD was incubated for 2 days prior to analysis. For differentiation studies, 25 µM MβCD and 4 µM FAK inhibitor 14 (Sigma-Aldrich) were used. MβCD was incubated until day 14 and was exchanged every 3 d. FAK inhibitor 14 was incubated until day 2 and then was exchanged for fresh growth medium.
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