Cell viability was evaluated through the MTT assay. Briefly, SH-SY5Y cells were treated with increasing concentrations of Aβ1-42 oligomers and fibrils (from 1 to 10 µM) in DMEM supplemented with 1% FCS (both from Euroclone, Italy) v/v for 48 h. The MTT assay was performed by incubating cells for 2 h at 37 °C with DMEM supplemented with 1% FCS and MTT [1 mg/mL]. After removing the supernatant, 300 µL of DMSO were added to each well to solubilize the newly-formed purple formazan. An aliquot of the suspension was finally collected and the absorbance was read at 570 nm. Cell viability was expressed as the percentage compared to the basal condition.
Dulbecco modified eagle medium (dmem)
Manufactured by Euroclone
Sourced in Italy, United States, United Kingdom
DMEM (Dulbecco's Modified Eagle Medium) is a cell culture medium formulation designed to support the growth and maintenance of various cell types. It provides the necessary nutrients, salts, and other components to sustain cell proliferation and viability in a laboratory setting.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Euroclone (90 mentions)
Streptomycin, by Euroclone (46 mentions)
Penicillin, by Euroclone (45 mentions)
L glutamine, by Euroclone (42 mentions)
Penicillin streptomycin, by Euroclone (33 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (33 mentions)
Rpmi 1640, by Euroclone (16 mentions)
Glutamine, by Euroclone (14 mentions)
Penicillin, by Merck Group (12 mentions)
Streptomycin, by Merck Group (12 mentions)
Fetal bovine serum (fbs), by Merck Group (12 mentions)
Rpmi 1640 medium, by Euroclone (10 mentions)
L glutamine, by Merck Group (10 mentions)
Trypsin, by Euroclone (8 mentions)
Dulbecco modified eagle medium (dmem), by Merck Group (7 mentions)
Penicillin, by Thermo Fisher Scientific (7 mentions)
Mcf 7, by American Type Culture Collection (6 mentions)
Sodium pyruvate, by Euroclone (6 mentions)
Hct116, by American Type Culture Collection (5 mentions)
Roswell park memorial institute (rpmi), by Euroclone (5 mentions)
283 protocols using dulbecco modified eagle medium (dmem)
1
Amyloid-beta oligomers and fibrils cytotoxicity
2
Cytotoxicity Assay of Novel Complexes
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A2780 (ovarian carcinoma), A549 (lung carcinoma) and MDA-MB-231 (breast carcinoma) cells (all obtained from ICLC, Genova, Italy) were grown as monolayers in Roswell Park Memorial Institute (RPMI 1640) or Dulbecco’s Modified Eagle’s Medium (DMEM) media (EuroClone, Pero, Italy) supplemented with 10% fetal bovinum serum (FBS) (Euroclone), antibiotics (EuroClone), and non-essential amino-acids (only DMEM, EuroClone). For the assay, cells plated into flat-bottomed 96-well microtiter plates were treated after 6–8 h with the complexes (five 1:5 scalar solutions, 20 µL, starting from 1 µM concentration). Seventy-two hours later, cells were analysed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium Bromide (MTT) assay as described elsewhere [33 (link)].
IC50 values were calculated from the analysis of single concentration–response curves. Final values are the mean of 4–12 experiments.
IC50 values were calculated from the analysis of single concentration–response curves. Final values are the mean of 4–12 experiments.
3
Primary Fibroblast Isolation from Skin
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Primary skin fibroblast cell lines were isolated from skin biopsies as follows. Skin specimens were washed with 70% ethanol and physiological solution and incubated with 2 mg/mL Dispase II (Merck KGaA, Darmstadt, Germany) overnight at 4 °C. Then, the dermis was separated using sterile tweezers, cut into 2–4 mm pieces and plated on a 6-well microplate. A squared sterile glass was put above and 1 mL of high glucose Dulbecco’s modified Eagle’s medium, DMEM (Euroclone, Milan, Italy) supplemented with 20% fetal bovine serum, FBS (Euroclone, Milan, Italy) was added to each well. After 3 weeks, dermis and glasses were removed and fibroblasts were detached with 0.25% Trypsin and 0.02% EDTA (Euroclone, Milan, Italy) and plated in 25 cm2 flasks. Cells were then cultured in DMEM supplemented with 15% (v/v) FBS, 100 U/mL penicillin, 100 mg/mL streptomycin (Euroclone, Milan, Italy) and 2 mM L-glutamine (Euroclone, Milan, Italy) and maintained at 37 °C under humidified conditions and 5% CO2. Cells were sub-cultured twice weekly, detached with Accutase (Euroclone, Milan, Italy) and centrifuged at 500× g for 10 min at room temperature. Cells were used for proteomics analysis at passage number lower than 10. Cell pellets were collected, washed in PBS, frozen in liquid nitrogen and stored at −80 °C.
4
Establishment and Maintenance of MPM Cell Lines
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Normal human mesothelial cells Met-5A and MPM cell lines ZL-55, REN, and MSTO were obtained from American Type Culture Collection (ATCC, Manassas, VA, USA). MPM cell lines Mero-14, Mero-41, and Mero-95 were obtained from European Collection of Authenticated Cell Culture (ECACC, Porton Down, UK). Met-5A cells were grown in Medium 199 supplemented with 10% FBS, 3.3 nM EGF, 400 nM hydrocortisone, and 870 nM zinc-free bovine insulin (all from Gibco, Carlsbad, CA, USA). Mero-14, Mero-41, and Mero-95 cells were grown in HAMS F10; ZL-55 and MSTO cells were grown in a 1:1 mixture of DMEM and Ham’s F-12; REN cells were grown in DMEM (all from Euroclone S.p.A., Milan, Italy). All MPM cells were maintained in medium supplemented with 10–15% heat-inactivated FBS, 2mM L-glutamine, 100 U/mL penicillin, and 100 U/mL streptomycin (all from Euroclone S.p.A., Milan, Italy). Cells were kept at 37 °C in a constant humidified 5% CO2 atmosphere.
5
In vitro model of intracerebral hemorrhage
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Brain microvascular endothelial cells (BMVECs) obtained from the Institute of Hematology, Chinese Academy of Medical Sciences, were used to establish a model of ICH in vitro. Cells were cultured in glucose- and serum-free dulbecco’s modified eagle medium (DMEM; EuroClone, Milan, Italy) under 5% CO2 and 95% N2 for 10 min. The cells were then cultured with hemin (10 μM, Sigma, New Jersey, USA) and maintained for 2 h in an anaerobic chamber filled with 5% CO2 and 95% N2 and restored back to normal culture conditions for oxygen and glucose deprivation (OGD) termination [23 (link)].
Transfections [23 (link)] were performed when cell confluence reached approximately 60%. The H19 inhibitor plasmid, miR-106b-5p mimic/inhibitor plasmid, OE-ACSL4 vector, and their negative controls (all obtained from Sangon, Shanghai, China) were diluted to a final concentration of 20 μM, and then 5 μl Lipofectamine® 3000 reagent (Thermo Fisher Scientific, Califonia, USA) were combined with 5 μl transfection plasmids, and incubated for 20 min at room temperature under the guidance of the manufacturer’s instruction. After 6 h of transfection, the solution was replaced with DMEM (EuroClone, Milan, Italy).
Transfections [23 (link)] were performed when cell confluence reached approximately 60%. The H19 inhibitor plasmid, miR-106b-5p mimic/inhibitor plasmid, OE-ACSL4 vector, and their negative controls (all obtained from Sangon, Shanghai, China) were diluted to a final concentration of 20 μM, and then 5 μl Lipofectamine® 3000 reagent (Thermo Fisher Scientific, Califonia, USA) were combined with 5 μl transfection plasmids, and incubated for 20 min at room temperature under the guidance of the manufacturer’s instruction. After 6 h of transfection, the solution was replaced with DMEM (EuroClone, Milan, Italy).
6
Isolation and Culture of Mammary Carcinoma Cell Lines
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The stromal cell line A17 was isolated from a murine model of mammary carcinoma induced by the overexpression of HER-2/neu transgene in the epithelial compartment of mammary glands in FVB mice, line 23325 (link). A17 cells were cultured in high-glucose Dulbecco’s Modified Eagle Medium (DMEM - Euroclone, Pero, Italy), supplemented with 20% fetal bovine serum (Gibco - Hyclone, South Logan, UT, USA), 1% penicillin-streptomycin and 1% glutamine.
WI38, NIH-3T3 and MCF-7 cell lines were kindly provided by Prof. Pier Giuseppe Pelicci (European Institute of Oncology - Milan, Italy, 2016) and cultured in high-glucose Dulbecco’s Modified Eagle Medium (DMEM - Euroclone, Pero, Italy), supplemented with 10% fetal bovine serum (Gibco - Hyclone, South Logan, UT, USA), 1% penicillin-streptomycin and 1% glutamine.
All the cell lines were grown in a humidified atmosphere at 37 °C and 5% CO2 as previously described61 (link)62 (link).
WI38, NIH-3T3 and MCF-7 cell lines were kindly provided by Prof. Pier Giuseppe Pelicci (European Institute of Oncology - Milan, Italy, 2016) and cultured in high-glucose Dulbecco’s Modified Eagle Medium (DMEM - Euroclone, Pero, Italy), supplemented with 10% fetal bovine serum (Gibco - Hyclone, South Logan, UT, USA), 1% penicillin-streptomycin and 1% glutamine.
All the cell lines were grown in a humidified atmosphere at 37 °C and 5% CO2 as previously described61 (link)62 (link).
7
Myotube Differentiation from Satellite Cells
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Myotubes were obtained after satellite cells differentiation. Satellite cells (SC) were prepared from glycolytic muscles following a standardised, automated tissue dissociation protocol with a gentleMACS™ Octo Dissociator with Heaters (Miltenyi Biotec, Bergisch Gladbach, Germany) and magnetic depletion as previously reported [21 (link)].
To generate myotubes, SC were cultured on Matrigel-coated (BD Biosciences, San Jose, CA, USA) coverslips in DMEM (EuroClone, Pero, Milan) supplemented with 20% foetal bovine serum (EuroClone), 3% chick embryo extract (custom made), 10 ng/ml basic fibroblast growth factor (PeproTech, London, UK) and 1% penicillin-streptomycin (EuroClone) at 37 °C with 5% CO2 for two days to reach the sufficient cells density to promote myogenic differentiation. Myogenic differentiation was induced in DMEM supplemented with 2% horse serum (EuroClone) and 1% penicillin-streptomycin (EuroClone) and after 48 h myotubes were analysed.
For some experiments, myotubes after 24 h in differentiation medium were supplemented with Mdivi-1 1 µM [22 (link)] in DMSO and analysed after 24 h.
To generate myotubes, SC were cultured on Matrigel-coated (BD Biosciences, San Jose, CA, USA) coverslips in DMEM (EuroClone, Pero, Milan) supplemented with 20% foetal bovine serum (EuroClone), 3% chick embryo extract (custom made), 10 ng/ml basic fibroblast growth factor (PeproTech, London, UK) and 1% penicillin-streptomycin (EuroClone) at 37 °C with 5% CO2 for two days to reach the sufficient cells density to promote myogenic differentiation. Myogenic differentiation was induced in DMEM supplemented with 2% horse serum (EuroClone) and 1% penicillin-streptomycin (EuroClone) and after 48 h myotubes were analysed.
For some experiments, myotubes after 24 h in differentiation medium were supplemented with Mdivi-1 1 µM [22 (link)] in DMSO and analysed after 24 h.
8
Ovarian Cancer Cell Line Cultivation
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Two ovarian cancer cell lines (OC316 and OVCAR3), representative of highly glycolytic cells [19 (link),20 (link)], were used in this study. OC316 cells were provided by S. Ferrini (IST, Genoa, Italy) and OVCAR3 cells were provided by S. Canevari (INT, Milan, Italy). OC316 and OVCAR3 were cultured in RPMI 1640 (Euroclone, Milan, Italy) supplemented with 10% fetal calf serum (FCS, Life Technologies, Paisley, UK), 1% HEPES (10mmol/L, Cambrex Bioscence, East Rutherford, NJ, USA), 1% Sodium Pyruvate (1 mmol/L), 1% L-glutamine (2 mmol/L), and 1% antibiotic–antimycotic mix 100× (Gibco–Thermo Fisher Scientific, Waltham, MA, USA). The human embryonic kidney epithelium cell line 293T was purchased from ATCC and cultured in Dulbecco modified Eagle medium (Euroclone), supplemented with 10% FCS, 10 mM HEPES and 1% of antibiotic–antimycotic mix (Thermo Fisher Scientific) and used to produce lentiviral vectors. Cultures were maintained at 37 °C in humidified 5% CO2/95% air atmosphere.
9
Cell Line Authentication and Culturing
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BJ-hTERT, BJ-hTERT/HRASG12V, BJ-hTERT/myrAKT1, IMR90, SK-LMS-1, SK-LMS-1HDAC4-/-HDAC4PAM-ER, were previously described [8 (link)]. Parental BJ, IMR90 and SK-LMS-1 have been authenticated by STR DNA Profiling (MWG Eurofins, Germany). BJ-TERT fibroblasts were grown in Earle’s salts minimal essential medium (Euroclone, Milan, Italy) completed with nonessential amino acids (Sigma-Aldrich St. Louis, MO, USA). IMR90 and SK-LMS-1 cells were cultured in Dulbecco modified Eagle medium (Euroclone, Milan, Italy). All mediums were supplemented with 10% of fetal bovine (FBS), L-glutamine 2 mM; penicillin 100 U/mL, and streptomycin, 100 μg/mL; (Euroclone). All cell lines were routinely tested from Mycoplasma contamination.
10
Isolation of Umbilical Cord-Derived MSCs
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Three human umbilical cords were obtained from women with healthy pregnancies during caesarean deliveries at the end of gestation after signing informed consents. Umbilical cords (UCs) were collected in phosphate-buffered saline (PBS) (Euroclone, Italy) supplemented with antibiotics, 100 U/ml penicillin and 100 μg/ml streptomycin (Euroclone, Italy) and transported to the laboratory. UCs were washed with PBS under a sterile laminar flow cell culture hood and cut into 5 cm2 segments. The segments were cut longitudinally, and blood vessels were removed, and segments transferred to 25 cm2 flasks and incubated at 37°C in a humidified atmosphere with 5% CO2 in DMEM (Euroclone, Italy) supplemented with either 10% FBS (Euroclone, Italy) or 10% CBS. Flasks were left undisturbed for 7 days, after which the medium was changed for the first time and later was changed every 3~4 days. After 2 weeks, the UC explants were removed and the adherent cells were allowed to expand until they reach about 80% confluence. Morphology of isolated and expanded MSC cells was assessed using inverted microscope IX50 (Olympus, Japan) during the cell culture period.
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