L-α-Phosphatidylcholine (PC) (Sigma Aldrich, St. Louis, MO, USA) and L-α-phosphatidic acid (PA) (Sigma Aldrich) were mixed to form unilamellar liposomes at a 2 : 1 molar ratio. Liposomes were obtained as described previously [4 (link)]. Briefly, PC and PA were dissolved in diethylether (JT Baker, Phillipsburg, NJ, USA), TS buffer (Tris-HCl) (Sigma Aldrich) was added, and the mixture was subjected to sonication cycles, diethylether elimination, and 0.45 μm filtration. To induce the formation of NPA in liposomes, liposomes were incubated with 3 mM chlorpromazine (CPZ) (Sigma Aldrich), 8 mM promazine (PZ) (Sigma Aldrich), or 5 mM MnCl2 (Mn2+) (Sigma Aldrich), respectively, for 30 min at 37°C. The presence of NPA in liposomes was detected by flow cytometry through a previously reported method [4 (link), 5 (link)], where liposome complexity is affected by the presence of NPA. FACSCalibur equipment (Becton Dickinson, San Jose, CA, USA) and FlowJo software (FlowJo, LLC, Ashland, Ore, USA) were used for data analysis.
Chlorpromazine cpz
Manufactured by Merck Group
Sourced in United States
Chlorpromazine (CPZ) is a chemical compound commonly used as a laboratory reagent. It is a phenothiazine derivative with the molecular formula C17H19ClN2S. CPZ serves as a standard reference material for various analytical and research applications.
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Lab products found in correlation
Amiloride, by Merck Group (3 mentions)
Genistein, by Merck Group (2 mentions)
Filipin, by Merck Group (2 mentions)
Wortmannin, by Merck Group (2 mentions)
Hepes, by Merck Group (2 mentions)
Acetonitrile, by Thermo Fisher Scientific (2 mentions)
Methanol, by Thermo Fisher Scientific (2 mentions)
Rpmi 1640 media, by Thermo Fisher Scientific (2 mentions)
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (2 mentions)
Facscalibur equipment, by BD (1 mentions)
L α phosphatidylcholine pc, by Merck Group (1 mentions)
Diethyl ether, by Merck Group (1 mentions)
Diethyl ether, by Avantor (1 mentions)
Monodansylcadaverine mdc, by Merck Group (1 mentions)
N acetylcysteine nac, by Merck Group (1 mentions)
Hepg2, by American Type Culture Collection (1 mentions)
Dmem high glucose media, by Merck Group (1 mentions)
3 methyladenine 3 ma, by Merck Group (1 mentions)
22 protocols using chlorpromazine cpz
1
Liposomal Phospholipid Transformation
2
Drugged AML-12 Cell Invasion Assay
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Invasion assays on an AML-12 cell line were performed in the presence of different inhibitors. AML-12 cells were pre-treated with drugs or their mock: amiloride (Sigma) at 1 mM for 30 min in culture medium, chlorpromazine CPZ (Sigma) at 10 μg/mL for 1 h in culture medium, monodansylcadaverine MDC (Sigma) at 300 μM for 1 h in DMSO, filipin (Sigma) at 5 μg/mL for 30 min in ethanol or genistein (Sigma) at 200 μM, for 30 min in DMSO. The dilution-effect of each drug was also performed. Viability of the bacteria was checked in the presence of all the dilutions of the drugs used. Bacteria (MOI = 10) diluted in the presence of the different drugs were deposited on cells for 1.5 h. Gentamicin was added for 1.5 h (using the same procedure as for the cell invasion assays). The number of internalized bacteria was determined and expressed in relation to values obtained for cells treated with control-diluted reagent arbitrarily set at 100%. Data are presented as the mean ± SEM. in duplicate and were repeated at least twice for each strain.
3
Cell Line Authentication and Treatment
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The human normal hepatocyte cell line MIHA and the HCC cell line SNU475 were kindly gifted to us by Professor Suk Woo Nam (The Catholic University, Seoul, Korea) and the HCC cell line HepG2 was purchased from ATCC (Manassas, VA, USA). All cell lines used in this study were grown in Roswell Park Memorial Institute (RPMI)-1640 or Dulbecco’s modified Eagle’s medium (DMEM)-high glucose media (Sigma-Aldrich, St. Louis, MO, USA) supplemented with 10% fetal bovine serum (FBS) (Hyclone, Logan, UT, USA) and 1% penicillin-streptomycin (Invitrogen, Carlsbad, CA, USA). The cells were incubated in humidified conditions with 5% CO2 at 37 °C. Mycoplasma contamination of all cell lines was tested using BioMycoX® Mycoplasma PCR Detection Kit (Cellsafe, Suwon, Korea) and short tandem repeat (STR) profiling was performed for authentication.
Valproic acid sodium salt (VPA) (Figure 1 A(i)), doxorubicin hydrochloride (DOX) (Figure 1 A(ii)), sodium butyrate, N-acetylcysteine (NAC), 3-methyladenine (3-MA), chlorpromazine (CPZ), methyl-β-cyclodextrin (MβCD), LY 294002 (LY), Hoechst 33258, and acridine orange hydrochloride hydrate (AO) were acquired from Sigma-Aldrich. 2′,7′-Dichlorodihydrofluorescein diacetate (H2DCFDA) was purchased from Molecular Probes™ (Eugene, OR, USA).
Valproic acid sodium salt (VPA) (
4
Aptamer-based Detection of SGIV Infection
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The aptamer Q5 was generated against SGIV-infected cells with Cell-SELEX in our previous study (Li et al., 2015 (link)). Aptamer Q5 was synthesized by Life Technologies, and the 5′ end of aptamer Q5 was labeled fluorescently with Cy5 (Cy5-Q5). Chlorpromazine (CPZ), methyl-β-cyclodextrin (MβCD), nystatin, genistein, dynasore, (N-ethyl-N-isopropyl)-amiloride (EIPA), ML-7, NSC23766, rottlerin, IPA-3, chloroquine (CQ), ammonia chloride (NH4Cl), cytochalasin D (cytoD), and nocodazole were purchased from Sigma-Aldrich (St. Louis, MO, United States). All reagents were stored and dissolved to specific concentrations according to the manufacturer’s instructions.
5
Investigating VLP Uptake Modulation
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hCMEC/D3 cells and iPSC-derived astrocytes were seeded with an equal density of 40 000 cells per cm2 on 96-well plates in order to evaluate the influence of 5HT2A receptor (5HT2AR) on VLP uptake. Each of them was first pre-treated for 1 h with rabbit anti-human 5HT2AR polyclonal antibody (1 : 200, Abcam) at 0.185 and 1.85 μg mL−1, or without the antibody. After removal of the antibody, the cells were washed with PBS and treated with 31.5 μg mL−1 VLPs in culture media for 24 h. For inhibition of clathrin-mediated endocytosis, chlorpromazine (CPZ) (Sigma-Aldrich) or Pitstop2 inhibitor (Abcam) was used respectively. CPZ was prepared with cell media before exposed to the cells for 1 h at concentrations of 1 and 100 μM, or without CPZ; in parallel, Pitstop2 inhibitor was dissolved in DMSO before diluted in cell media for cell treatment of 30 min at 30 μM, similar to a previous report.31 (link) Pitstop2 negative control (Abcam) featured by its highly related structure to Pitstop2 inhibitor was also applied at 30 μM, together with DMSO only and untreated controls. After the pre-treatment, CPZ and Pitstop2 were removed and rinsed. The cells were subsequently incubated with 31.5 μg mL−1 VLPs for 1.5 h and stained for anti-VP1 immunocytochemistry. The analysis was performed on Arrayscan HCA studio as described above.
6
Endocytic Pathways for Gene Delivery
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HEK293-RFP cells at ~70% confluency were incubated with one of the following endocytosis inhibitors for 1 h: NaN3 (Sigma), 5-(N-ethyl-N-isopropyl)amiloride (EIPA, Santa Cruz Biotechnology), Dynasore (Abcam), Chlorpromazine (CPZ, Sigma), Methyl-β-cyclodextrin (MBCD, Sigma), Wortmannin (Sigma). Then, liposomes containing either (-30)GFP-Cre or ProTα-Cre were delivered and incubated at 37 °C with 5% CO2 for 4 h. The cells were washed three times with PBS containing heparin (Stem Cell Technologies) at 20 µg/ml. The cells were further recovered for 2 day, and the cells were analyzed by the CytoFlex flow cytometer (Beckman Coulter).
7
Cellular Uptake Mechanism of Abraxane
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Abx was labeled with FITC using fluorescein-EX protein labeling kit (Thermo Fisher). A 0.5 mL solution of 2 mg/mL abraxane was mixed with 50 µl of 1 M bicarbonate and incubated with the reactive dye for 1 h at room temperature. The labeled Abx was purified from free dye using a spin column (MW > 20 kDa). Cells (1 × 105 cells/well of a four-well chamber) were incubated with 1% bovine serum albumin at 24°C for 30 min and then with 5 μg/mL of FITC-labeled Abx at 37 °C for 1 h. Cells were fixed with 4% paraformaldehyde followed by nuclear staining with 4', 6-diamidino-2-phenylindole (DAPI) after washing and observed under a confocal microscope (Nanoscope, Daejeon, Korea), or cells were analyzed using Attune NxT flow cytometer (Thermo Fisher). For the internalization inhibition, cells were incubated with 50 μM of 5-(n-ethyl-n-isopropyl) amiloride (EIPA, Thermo Fisher), 50 μM of chlorpromazine (CPZ, Sigma-Aldrich), and 100 μM of anti-IL4Rα blocking antibody or anti-IgG antibody (R&D Systems, Minneapolis, MN) for 30 min prior to incubation with FITC-labeled Abx.
8
Lipid-based Nanoparticle Formulations for Cellular Studies
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Ptx, trimyristin (Tm), verapamil (Ver), genistein (Gen), chlorpromazine (Cpz), and n-hexyl-hydroxybenzoate were purchased from Sigma-Aldrich Co. (St Louis, MO, USA). Egg L-α-phosphatidylcholine (PC) and DSPE– methyl(polyethylene glycol)-2000 (mPEG2,000) were purchased from Avanti Polar Lipids (Alabaster, AL, USA). Rhodamine 123 (Rho), DAPI, Bodipy FL C5-lactosylceramide complexed with BSA (LacCer), human serum transferrin (hTf), Alexa Fluor 488 conjugate, PBS, and other cell-culture reagents, including RPMI 1640 media, were from Thermo Fisher Scientific (Waltham, MA, USA). Acetonitrile, tert-butyl methyl ether, and methanol were of high-performance liquid chromatography (HPLC) grade and purchased from Thermo Fisher Scientific. All other reagents were of analytical grade and used without further purification.
9
Investigating BSEP Downregulation in Spheroids
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On day 7 post seeding, coculture spheroids were treated with different concentrations of Chlorpromazine (CPZ, Sigma-Aldrich, Switzerland) and Troglitazone (LKT Labs, USA) for 24 h. Bile salt export pump (BSEP) downregulation was measured from pooled spheroids via qRT-PCR.
10
Evaluating MkH's Effect on Efflux Pumps
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The effect of MkH on efflux pumps was evaluated in E. coli AG100TET strain, following the protocol described by Paixão et al. (2009) involving accumulation and efflux assays using 1 mg/L of ethidium bromide (EtBr; Sigma-Aldrich, Munich, Germany). Bacteria treated with 20 mg/L of chlorpromazine (CPZ; Sigma-Aldrich Química SA Madrid, Spain), an efflux pump inhibitor, was used as positive control. EtBr is a common efflux pump substrate; under normal conditions EtBr enters the bacteria and is rejected to outside by efflux pump action. In case these pumps are blocked EtBr accumulates inside the bacteria, increasing its fluorescence [37 (link)].
In this assay four concentrations of MkH (20%, 30%, 40% and 50%) were tested. Bacterial suspensions were incubated for 60 min at 37 °C and 180 rpm in darkness. The fluorescence intensity (FI) was measured at FL3. MIF was used to evaluate treatment differences.
In this assay four concentrations of MkH (20%, 30%, 40% and 50%) were tested. Bacterial suspensions were incubated for 60 min at 37 °C and 180 rpm in darkness. The fluorescence intensity (FI) was measured at FL3. MIF was used to evaluate treatment differences.
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