This research was financially supported under Grant No. 398617 by the Isfahan University of Medical Sciences. The Iranian reference strain of Leishmania major (MHRO/IR/75/ER) was provided by the Department of Parasitology at the Isfahan University of Medical Sciences. A frozen vial including parasite was removed from the nitrogen tank. A drop of parasite was examined using a light microscope. After confirming the presence of live cells, the parasite vial was rinsed in RPMI 1640 (Bioidea, USA) under sterile condition in several steps to remove the DMSO effect. It was then transferred to the pre-prepared Novy-MacNeal-Nicolle medium (NNN), a two-phase culture medium. The samples were placed in refrigerator incubator at 24–25°C temperature while the growth of promastigotes was checked every 2 hours. After growing the promastigotes in the liquid phase up to a mass growth in the logarithmic phase for use in animal infection, the parasite was added in RPMI liquid and single-phase culture medium enriched with 20% FCS and antibiotics penicillin G (100 IU/ml) and streptomycin (100 μg/ml). The tubes were re-incubated at 25°C to multiply the parasites. Finally, promastigotes were examined under a microscope and the number of parasite cells was counted by a neobar slide.
Rpmi 1640
Manufactured by Bioidea
Sourced in United States
RPMI-1640 is a widely used cell culture medium formulated to support the growth of a variety of cell types, including human and animal cell lines. It provides essential nutrients and growth factors necessary for cell proliferation and maintenance in in vitro cell culture applications.
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Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (5 mentions)
Fetal bovine serum (fbs), by Bioidea (5 mentions)
Amphotericin b, by Merck Group (3 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (3 mentions)
Penicillin streptomycin, by Bioidea (3 mentions)
Trypsin edta, by Bioidea (2 mentions)
Streptomycin, by Bioidea (2 mentions)
Penicillin, by Bioidea (2 mentions)
Penicillin streptomycin antibiotic, by Bioidea (1 mentions)
Lipopolysaccharide lps from escherichia coli 0127 b8, by Merck Group (1 mentions)
Anti rabbit and anti mouse igg hrp linked antibodies, by Cell Signaling Technology (1 mentions)
Polyvinylidene uoride pvdf membrane, by Bio-Rad (1 mentions)
Nisin, by Merck Group (1 mentions)
Annexin 5 fitc kit, by Abcam (1 mentions)
Methyl thiazolyl tetrazolium (mtt), by Merck Group (1 mentions)
Trypsin edta 1x, by Thermo Fisher Scientific (1 mentions)
Paclitaxel, by Merck Group (1 mentions)
Taxol, by Merck Group (1 mentions)
Tween 20, by Merck Group (1 mentions)
Posaconazole, by Merck Group (1 mentions)
18 protocols using rpmi 1640
1
Cultivation and Enumeration of Leishmania major
2
Caco-2 Cell Culture for Differentiation Studies
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Caco-2 ATCC HTB-37 was provided by the Pasteur Institute of Iran (Tehran, Iran). Cells were grown in RPMI 1640 (BioIdea, Iran) supplemented with 20% fetal bovine serum (FBS) (Gibco, USA), 1% (v/v) penicillin-streptomycin antibiotic (10,000 IU/mL and 10,000 μg/mL, respectively; BioIdea, Iran) and amphotericin B (50 mg/10 mL) (Sigma, USA). The Cells were incubated at 37 °C in 5% CO2. For subsequent assays, cell monolayers were prepared in 96-well tissue culture plates by seeding 3 × 104 cells/well and incubated for 48 h to reach confluence. In Caco-2 cells, experiments were carried out after cells were differentiated (15 days post-confluence) [17 (link)].
3
Macrophage Activation Assay with Fluoxetine
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Fluoxetine hydrochloride was purchased from Dr. Abidi Pharmaceuticals (Iran). The RAW264.7 mouse macrophage cell line was purchased from the Cell Bank of Pasteur Institute of Iran (Iran). Dimethyl sulfoxide (DMSO) and lipopolysaccharide (LPS from Escherichia coli 0127:B8) were obtained from Sigma-Aldrich (USA). RPMI-1640, Dulbecco's modified eagle medium (DMEM), and fetal bovine serum (FBS) were obtained from Bioidea (Iran). Collagen- and fibronectin-coated dishes were purchased from Padgin Teb Company (Iran).
4
Investigating Cell Apoptosis in PC12 Cells
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Fluorescent probe 2,7-dichloro uorescein diacetate (DCF-DA) and (4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium (MTT) were purchased from Sigma. RPMI 1640 was purchased from Bioidea Company and FBS was purchased from Gibco. Rabbit monoclonal anti-serums against procaspase-3, caspase-3cleaved, Bcl-2, caspase-8, and ß-actin, rabbit polyclonal anti-serum against Bax, and anti-mouse and antirabbit IgG HRP-linked antibodies were purchased from Cell signaling. Cadmium and minocycline were purchased from Tinab Shimi. Polyvinylidene uoride (PVDF) membrane was purchased from Bio-Rad.
Cell culture PC12 (pheochromocytoma cell line of rat) cells were obtained from the Pasteur Institute of Iran (Tehran). Cells were cultured in RPMI-1640 medium and incubated at 37˚C with 5% CO 2 . The medium contained 10% fetal bovine serum and 5% antibiotic mixture (100 U/mL penicillin and 100 µg/mL streptomycin) and was changed every 2-3 days.
Cell culture PC12 (pheochromocytoma cell line of rat) cells were obtained from the Pasteur Institute of Iran (Tehran). Cells were cultured in RPMI-1640 medium and incubated at 37˚C with 5% CO 2 . The medium contained 10% fetal bovine serum and 5% antibiotic mixture (100 U/mL penicillin and 100 µg/mL streptomycin) and was changed every 2-3 days.
5
Melanoma Cell Line B16F10 Cytotoxicity Assay
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Melanoma cell line, B16F10, maintained at 37 °C in a humidified atmosphere (90%) containing 5% CO2. Cells were cultured in RPMI-1640 (Bioidea, Iran) with 10% (v/v) FBS, 100 IU/mL penicillin, and 100 μg/mL streptomycin. The stock solution of the P. atlantica subsp. mutica was prepared at 50 μg/mL in DMSO and kept at -40 °C. Kojic acid (2 and 4 mM) was used as positive control in all experiments. Cells were cultured in 96-well plates in a density of 105 cells/mL. The activity of the extract in each experiment was calculated using the following equation:
6
Nisin Concentration Preparation in RPMI1640
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7
Cytotoxic Activity of Taxol on A2780cp Cells
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A2780cp cell line (Human epithelial ovarian carcinoma) was purchased from NCBI (National Cell Bank of Iran). Trypsin/EDTA (1X) and fetal bovine serum were provided from Gibco (USA), RPMI-1640 and Trypan blue were purchased from Bio idea (Iran), and penicillin/streptomycin and phosphate buffer saline were purchased from PAA (Austria). PI (Propodium Iodide), DAPI (4′, 6-diamidino-2-phenylindole dihydrochloride) kit and MTT [3-(4, 5-dimethylthiozol-2-il) 2, 5 di phenyl tetrazolium bromide] and acridine orange/propodium iodide were purchased from Sigma (USA). Annexin V-FITC kit and Caspase-9 assay and Caspase-3 assay kit were prepared from Abcam (UK). Taxol or paclitaxel was purchased from Sigma (USA).
8
Antifungal Susceptibility Testing Protocol
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Twofold serial dilutions of antifungals including, luliconazole (APIChem Technology, China) (from 0.00012 to 0.25 μg/mL), amphotericin B (Sigma - Aldrich, Germany) (from 0.125 to 16 μg/mL), voriconazole (Sigma- Aldrich, Germany) (from 0.0078 to 4 μg/mL), posaconazole (Sigma - Aldrich, Germany) (from 0.0312 to 4 μg/mL), and caspofungin (Sigma - Aldrich, Germany) (from 0.0078 to 1 μg/mL) were prepared in RPMI 1640 (Bio Idea, Iran). Antifungal susceptibility test was performed according to CLSI M38 A2 (32 ). A standard suspension (0.5 McFarland) of 48–72 hours cultures on SDA was prepared in sterile saline (0.85%) with 0.2% Tween 20 (Merck, Germany). Then, 100 μL of diluted suspension (1:50) and 100 μL of serial dilutions of each antifungal were added to each well of 96-well microplates. Micro-plates incubated at 35ºC for 24–72 hours and results were recorded as MIC or minimum effective concentration (MEC). Finally, MIC or MEC range, MIC50 or MEC50, MIC90 or MEC90 and MIC geometric (GM) or MECGM were calculated. CLSI or EUCAST have not been defined any clinical or epidemiologic breakpoints/cut-offs for amphotericin B, voriconazole, posaconazole, caspofungin and Aspergillus species. Strains susceptibility or resistance to each antifungals was evaluated according to commonly utilized breakpoints (Table 1 ) (33 –38 (link)).
9
Evaluation of Cell Viability and Insulin Assay
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RPMI-1640 and Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), Trypsin-EDTA and MTT (2, 3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide) were purchased from Bio-Idea Company (Tehran. Iran). BSA was purchased from Solarbio (Life Sciences). Penicillin–streptomycin was purchased from Gibco Life Technologies (Paisley, UK). Glucose oxidase was purchased from Pars Azmon Company (Tehran, Iran). The insulin radioimmunoassay kit was purchased from Mercodia. The protein assay reagent was obtained from Bio-Rad (Copenhagen, Denmark). The chemicals including sulfuric acid, ethanol, Na2So4, KOH, all were purchased from Merk (Germany).
10
Evaluating N. satureioides Cytotoxicity on B16F10 Melanoma Cells
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B16F10 melanoma cell line (Cat. No C540) was purchased from the Pasteur Institute of Iran (Tehran, Iran) and maintained at 37 °C in a humidified atmosphere (90%) containing 5% CO2. Cells were cultured in RPMI-1640 (Bioidea, Iran) with 10% (v/v) fetal bovine serum, 100 U/mL penicillin, and 100 μg/mL streptomycin. In order to evaluate the cytotoxic effect of different fractions of N. satureioides on cells, 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) cytotoxic assay was performed(13 (link)). To monitor cell viabilities, cells (2 × 104 cells per well) seeded in a 96-well plate overnight. The cells then exposed to different concentrations of fractions (5 – 60 μg/mL) for 24 h.
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