RPMI-1640 medium was obtained from ATCC (Manassas, VA, USA). Fetal bovine serum (FBS), phosphate buffered saline (PBS, 0.01 M, pH 7.4), trypsin, penicillin-streptomycin (pen/strep) solution, crystal violet, dimethylsulfoxide (DMSO), cytochalasin-B and RNAse were obtained from Sigma-Aldrich (St. Louis, MO, USA). The TrypLE™ Express Enzyme solution was obtained from Gibco, Invitrogen (UK). The (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulphophenyl)-2H-Tetrazolium)-CellTiter 96® AQueous One Solution Reagent (MTS) was obtained from Promega Corp. (Madison, WI, USA). The Giemsa dye, methanol, ethanol, acetic acid and propidium iodide (PI) were purchased from Merck (Darmstadt, Germany). The dihydrorhodamine 123 (DHR) and the Alexa Fluor® 488 Annexin V/PI kit were acquired from Molecular Probes (Eugene, OR, USA). The 10 mM stock solution of DHR was prepared in DMSO, aliquoted, and stored under nitrogen at −20 °C. The MnTnHex-2-PyP5+ (MnP) was synthetized as described previously [20 (link)] (charges are omitted for clarity throughout the manuscript).
Penicillin streptomycin pen strep solution
Manufactured by Merck Group
Sourced in United States, Germany, Italy
Penicillin-streptomycin (pen/strep) solution is a common antibiotic mixture used in cell culture applications. It is a sterile, liquid solution that contains the antibiotics penicillin and streptomycin.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Merck Group (3 mentions)
Trypsin, by Merck Group (2 mentions)
Propidium iodide, by Merck Group (2 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (2 mentions)
Methanol, by Merck Group (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (2 mentions)
Fetal bovine serum (fbs), by Euroclone (2 mentions)
Dulbecco s modified eagle medium, by Merck Group (2 mentions)
Crystal violet, by Merck Group (1 mentions)
Giemsa dye, by Merck Group (1 mentions)
Rpmi 1640 medium, by American Type Culture Collection (1 mentions)
Dihydrorhodamine 123 dhr, by Thermo Fisher Scientific (1 mentions)
Tryple express enzyme solution, by Thermo Fisher Scientific (1 mentions)
Cytochalasin b, by Merck Group (1 mentions)
Celltiter 96 aqueous one solution reagent, by Promega (1 mentions)
Acetic acid, by Merck Group (1 mentions)
Ethanol, by Merck Group (1 mentions)
Easyflask, by Thermo Fisher Scientific (1 mentions)
Panc 1 cell line, by American Type Culture Collection (1 mentions)
10 protocols using penicillin streptomycin pen strep solution
1
Cell Viability and Apoptosis Assays
2
Exosome Production from PANC-1 Cells
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PANC-1 cell line was purchased from American Type Culture Collection (Manassas, VA, USA). After thawing from dry ice, PANC-1 cells were cultured in Nunc™ EasYFlask™ cell culture flasks in 95% humidity and 5% CO2 at 37 °C. The base medium for the cells was Dulbecco's Modified Eagle's Medium (DMEM, Thermofisher) supplemented with 10% exosomes-depleted heat-inactivated fetal bovine serum (FBS, Thermofisher) and 1% Penicillin-Streptomycin (Pen Strep) solution (Sigma). Cells were maintained for at least 2 passages before seeding for exosome experiments and spheroid generation.
For exosome production from 2D cultured PANC-1 cells, 5 × 104 cells were seeded in 1 mL growth medium for a single well of a 24-well plate. After 24 h when cells reached approximately 70% confluency [23 (link)], cells were washed with PBS and fresh DMEM supplemented with exosomes-depleted FBS was replaced (Day 0). Medium was changed every other day. At Day 5, culture supernatant (1 mL) was collected and centrifuged at 300×g for 10 min at room temperature to remove cell debris.
For exosome production from 2D cultured PANC-1 cells, 5 × 104 cells were seeded in 1 mL growth medium for a single well of a 24-well plate. After 24 h when cells reached approximately 70% confluency [23 (link)], cells were washed with PBS and fresh DMEM supplemented with exosomes-depleted FBS was replaced (Day 0). Medium was changed every other day. At Day 5, culture supernatant (1 mL) was collected and centrifuged at 300×g for 10 min at room temperature to remove cell debris.
3
Ionic Liquid-Based Rutin Nanoparticles
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The reagents and solvents used for the synthesis of the ILs were choline hydroxide in methanol [Cho][OH]/MeOH 45% and methanol, both from Sigma-Aldrich (Saint Louis, MO, USA), also acetonitrile from VWR (Fontenay-sous-Bois, France) and the amino acids, L-phenylalanine and glycine, from PanReact AppliChem (Barcelona, Spain).
For the cytotoxicity studies, the following reagents were purchased from Sigma-Aldrich (Saint Louis, MO, USA), phosphate buffered saline (PBS; 0.01 M, pH 7.4), trypsin, penicillin–streptomycin (pen/strep) solution, thiazolyl blue tetrazolium bromide (MTT) and dimethyl sulfoxide (DMSO). Fetal bovine serum (FBS) and Dulbecco’s Modified Eagle’s Medium (DMEM) were from Biowest (Nuaillé, France). The propidium iodide (PI) was purchased from Merck (Darmstadt, Germany). All the rutin solutions had a final concentration of 0.5% (v/v) and were prepared in DMSO, for all assays. The solutions containing ionic liquids were all prepared in sterile water.
For the production of the nanoparticles, the dichloromethane and the polyvinyl alcohol (PVA) were obtained from Sigma-Aldrich (St. Louis, MO, USA). Corbion Purac (Amsterdam, The Netherlands) kindly supplied the Poly(lactic-co-glycolic acid) (PLGA) 50:50 with acidic termination (Purasorb® PDLG 5002A).
Rutin was obtained from Fagron, São Paulo, Brazil.
For the cytotoxicity studies, the following reagents were purchased from Sigma-Aldrich (Saint Louis, MO, USA), phosphate buffered saline (PBS; 0.01 M, pH 7.4), trypsin, penicillin–streptomycin (pen/strep) solution, thiazolyl blue tetrazolium bromide (MTT) and dimethyl sulfoxide (DMSO). Fetal bovine serum (FBS) and Dulbecco’s Modified Eagle’s Medium (DMEM) were from Biowest (Nuaillé, France). The propidium iodide (PI) was purchased from Merck (Darmstadt, Germany). All the rutin solutions had a final concentration of 0.5% (v/v) and were prepared in DMSO, for all assays. The solutions containing ionic liquids were all prepared in sterile water.
For the production of the nanoparticles, the dichloromethane and the polyvinyl alcohol (PVA) were obtained from Sigma-Aldrich (St. Louis, MO, USA). Corbion Purac (Amsterdam, The Netherlands) kindly supplied the Poly(lactic-co-glycolic acid) (PLGA) 50:50 with acidic termination (Purasorb® PDLG 5002A).
Rutin was obtained from Fagron, São Paulo, Brazil.
4
IFN-β treatment of HeLa and HT1080 cells
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HeLa (ATTC: CCL-2) and HT1080 (ATCC: CCL-121) cells were cultured as adherent cells at 37 °C with 5% CO2 in Dulbecco’s modified Eagle’s medium with high glucose and with sodium pyruvate (Life Technologies Europe BV, Nærum, Denmark) with added 10% sterile-filtered fetal bovine serum (FBS) (Sigma-Aldrich, Søborg, Denmark ) and 1% Penicillin-Streptomycin (Pen-Strep) solution (Sigma-Aldrich) at 37 °C and 5% CO2. The human HT1080 and HeLa cell lines used have the AG genotype and the AA genotype, respectively, regarding the SNP rs10774671 [9 (link)]. For interferon treatments, both HeLa and HT1080 cells were grown to ~80% confluence and were treated with 1000 U/mL IFN-β or left untreated for 24–48 h before analysis. Both cell-lines were tested and found mycoplasma free.
5
Phenolic Standards and Cell Culture Reagents
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Phenolic standards such as salicylic acid, syringic acid, ferulic acid, gallic acid, p-hydroxybenzoic acid, p-coumaric acid, protocatechuic acid, vanillic acid, rosmarinic acid, caffeic acid, ellagic acid, chlorogenic acid, catechin, luteolin-7-glucoside, luteolin, apigenin-7-O-glucoside, apigenin, quercetin, rutin, kaempferol, isorhamnetin, naringin, methyl paraben, and vanillin were purchased from Sigma-Aldrich (Darmstadt, Germany). Solvents and reactants such as acetonitrile, acetic acid, ethyl acetate methanol, N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA) pyridine, and thiazolyl blue tetrazolium bromide (MTT, cat. no. M5655) were obtained from Merck KGaA (Darmstadt, Germany). Dulbecco’s Modified Eagle’s Medium (DMEM), fetal bovine serum (FBS), and penicillin–streptomycin (pen–strep) solution were obtained from Millipore Co. (Merck KGaA, Darmstadt, Germany). Other cell culture reagents were purchased from Gibco (Thermo Fisher Scientific, Inc, Waltham, MA, USA).
6
Cytotoxicity Screening of Pharmaceutical Compounds
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Cell culture reagents (cell culture medium, phosphate-buffered saline (PBS), fetal bovine serum (FBS), and penicillin–streptomycin (pen–strep) solution) were purchased from Millipore Sigma (Merck KGaA, Darmstadt, Germany). Other cell culture reagents were bought from Gibco (Thermo Fisher Scientific, Inc, Waltham, MA, USA). Fluphenazine (cat. no. F4765), pyronaridine (cat. no. P0049), edaravone (cat. no. M70800), 5-FU (cat. no. F6627), and Thiazolyl Blue Tetrazolium Bromide (MTT, cat. no. M5655) were obtained from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany). Sertraline (cat. no. 14839), quetiapine (cat. no. 14152), and DOX (cat. no. 15007) were acquired from Cayman Chemical (Ann Arbor, MI, USA). PTX (cat. no. 1097) was obtained from Tocris Bioscience (Bristol, UK). Mefloquine (cat. no. sc-211784) was purchased from Santa Cruz Biotechnology (Dallas, TX, USA).
7
Biocompatibility Evaluation of Novel Biomaterials
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Biocompatibility (cytotoxicity) was evaluated using fibroblasts (normal human dermal fibroblasts, from juvenile foreskin from 2 to 5 passages, PromoCell, WVR, Italy).
Fibroblasts were grown in DMEM (Lonza, I), 10% v/v (FBS (EuroClone, Italy) and penicillin/streptomycin solution (pen/strep, 100 UI/100 μm/mL, Sigma Aldrich-Merck, Italy)
VHS (1.2 mg/mL), NF (0.1 mg/mL) and VHS-NF (VHS: 1.2 mg/mL and NF 0.1 mg/mL) were suspended/solubilized in cell culture medium and put in contact with the cells in suspension just after cell seeding in 96-well plate at seeding density of 35,000 cells/well. Fibroblasts were grown for 2 days. Fibroblast growth on tissue culture plastic was considered as standard growth (control).
At prefixed end point, cell growth was assessed by means of MTT test. Briefly, the medium was removed and 50 µL of MTT solution (Sigma Aldrich, Italy) at 2.5 mg/mL concentration in Hank’s Buffered Salt Solution pH 7.4 was added to cover each scaffold for 3 hrs. Subsequently, MTT solution was removed from each well, and the substrates were washed with 200 µL of PBS. After the removal of PBS, 100 µL of DMSO was put in each well, and the absorbance was assayed at 570 nm by means of an ELISA plate reader (Imark Absorbance Reader, Biorad, Italy), with a reference wavelength of 690 nm. Cell viability was expressed as optical density (OD).
Fibroblasts were grown in DMEM (Lonza, I), 10% v/v (FBS (EuroClone, Italy) and penicillin/streptomycin solution (pen/strep, 100 UI/100 μm/mL, Sigma Aldrich-Merck, Italy)
VHS (1.2 mg/mL), NF (0.1 mg/mL) and VHS-NF (VHS: 1.2 mg/mL and NF 0.1 mg/mL) were suspended/solubilized in cell culture medium and put in contact with the cells in suspension just after cell seeding in 96-well plate at seeding density of 35,000 cells/well. Fibroblasts were grown for 2 days. Fibroblast growth on tissue culture plastic was considered as standard growth (control).
At prefixed end point, cell growth was assessed by means of MTT test. Briefly, the medium was removed and 50 µL of MTT solution (Sigma Aldrich, Italy) at 2.5 mg/mL concentration in Hank’s Buffered Salt Solution pH 7.4 was added to cover each scaffold for 3 hrs. Subsequently, MTT solution was removed from each well, and the substrates were washed with 200 µL of PBS. After the removal of PBS, 100 µL of DMSO was put in each well, and the absorbance was assayed at 570 nm by means of an ELISA plate reader (Imark Absorbance Reader, Biorad, Italy), with a reference wavelength of 690 nm. Cell viability was expressed as optical density (OD).
8
Adhesion and Proliferation of Normal Human Dermal Fibroblasts
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Adhesion and proliferation assay was carried out using normal human dermal fibroblasts (NHDF) from juvenile foreskin (PromoCell, VWR, Milan, Italy) [9 (link),10 (link)]. Fibroblasts were grown in the presence of 150 µL Dulbecco’s modified Eagle medium (DMEM, Sigma-Aldrich, Milan, Italy) supplemented with 10% v/v fetal bovine serum (Euroclone, Milan, Italy), and with penicillin/streptomycin solution (pen/strep, 100 UI/100 μg/mL, Sigma-Aldrich, Merck, Milan, Italy), at 37 °C in a 5% CO2 atmosphere with 95% relative humidity. The 0.36 cm2 circular portion scaffolds were placed at the bottom of the wells in a 96-well plate (flat bottom, Cellstar©, Greiner bio-one, Frickenhausen, Germany). Fibroblasts were seeded onto the scaffolds at a seeding density of 35,000 cells/well and grown for 3 or 6 days. The cell growth without scaffolds (35,000 cells/well) was considered the standard growth (growth medium (GM)). After 3 or 6 days, the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assays were performed. The fibroblasts that adhered and grew onto the scaffolds (growth for 6 days) were fixed for 2 h at 4 °C, using 3% w/v of glutaraldehyde in Dulbecco’s phosphate buffered saline (PBS, Sigma-Aldrich, Milan, Italy) and analyzed by SEM and confocal laser scanning microscopy (CLSM), as described in the following paragraphs.
9
Cytotoxicity Assessment of Extracts in HaCaT Cells
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Cytotoxicity was tested on HaCaT non-tumor human keratinocytes. The cell line was cultured in DMEM medium (Dulbecco’s Modified Eagle Medium, Sigma-Aldrich) supplemented with 10% fetal bovine serum (Sigma-Aldrich) and 1% Pen/Strep (penicillin/streptomycin solution, 50 µg/mL—Sigma-Aldrich) at 37 °C, 95% humidity, and 5% CO2. Cells were washed with phosphate-buffered saline (PBS, Sigma Aldrich), trypsinized (0.25% Trypsin-0.53 mM EDTA, Thermo Scientific), and counted using Trypan Blue and a Burker–Turk counting chamber. Different concentrations of extracts (5–50 µL/mL) were incubated with the cells (which were 24 h pre-seeded at a density of 2 × 104 cells/well) in a final volume of 200 µL growth medium for 24 h (37 °C, 95% humidity, 5% CO2).
10
Cytotoxicity Evaluation of Biomaterials
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L929 fibroblasts (5 × 105 cells/well) were cultured in DMEM (Dulbecco’s Modified Eagle Medium, Sigma-Aldrich) [120 (link)] medium supplemented with 10% fetal bovine serum (Sigma-Aldrich) and 1% pen/strep (penicillin/streptomycin solution, Sigma Aldrich) for 24 h at 37 °C in 95% humidity with 5% CO2.
The samples were co-cultured with the fibroblasts for 24 h (37 °C, 95% humidity, 5% CO2). The MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) [121 ] assay was used to evaluate the cell viability and proliferation in the presence of the materials. Cells were incubated for 4 h with MTT reagent (Vybrant® MTT Cell Proliferation Assay Kit, V-13154) at 37 °C in 95% humidity with 5% CO2. After incubation, formazan crystals were solubilized with DMSO for 10 min at room temperature. The absorbance was measured at λ = 540 nm using the Multiskan FC apparatus (Life Technologies Holdings Pte. Ltd., a part of Thermo Fisher Scientific Inc., Singapore). The cell morphology was evaluated using an inverted Olympus IX73 microscope after 24 h of incubation with the biomaterials.
The samples were co-cultured with the fibroblasts for 24 h (37 °C, 95% humidity, 5% CO2). The MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) [121 ] assay was used to evaluate the cell viability and proliferation in the presence of the materials. Cells were incubated for 4 h with MTT reagent (Vybrant® MTT Cell Proliferation Assay Kit, V-13154) at 37 °C in 95% humidity with 5% CO2. After incubation, formazan crystals were solubilized with DMSO for 10 min at room temperature. The absorbance was measured at λ = 540 nm using the Multiskan FC apparatus (Life Technologies Holdings Pte. Ltd., a part of Thermo Fisher Scientific Inc., Singapore). The cell morphology was evaluated using an inverted Olympus IX73 microscope after 24 h of incubation with the biomaterials.
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