X-MAN™ HCT116 Parental and HCT116 PTEN-/- cells and HFF cells were seeded alone or with a 1:1 ratio in 35 mm plates (Falcon BD, Oxford, UK) in RPMI 1640 (Euroclone, Milan, Italy) supplemented with 10% inactivated FBS (Gibco, Life Technologies, California, USA), as previously described. After 24 h, culture media were replaced with serum-free DMEM (Euroclone, Milan, Italy) containing different drug concentrations alone or in combination. After 72 h of treatment, cells were suspended in ice-cold RPMI 1640 (Euroclone, Milan, Italy) supplemented with 10% inactivated FBS (Gibco, Life Technologies, California, USA). The total amount of cells was counted using the Thoma chamber and the percentage of CRC and HFF cells were analyzed by flow cytometry analysis that allows to differentiate between HFF cellular populations, fluorescently labeled in comparison to unlabeled X-MAN™ isogenic HCT116 cells.
Rpmi 1640
Manufactured by Euroclone
Sourced in Italy, United Kingdom, United States, Austria
RPMI 1640 is a cell culture medium formulation commonly used for the growth and maintenance of a variety of cell types, including human and animal cells. It provides the necessary nutrients and components to support cell proliferation and viability in in vitro cell culture applications.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Euroclone (49 mentions)
L glutamine, by Euroclone (38 mentions)
Streptomycin, by Euroclone (36 mentions)
Penicillin streptomycin, by Euroclone (36 mentions)
Penicillin, by Euroclone (34 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (19 mentions)
Glutamine, by Euroclone (17 mentions)
Dulbecco modified eagle medium (dmem), by Euroclone (16 mentions)
Fetal bovine serum (fbs), by Merck Group (11 mentions)
Sodium pyruvate, by Merck Group (10 mentions)
Non essential amino acids (neaa), by Euroclone (9 mentions)
Hepes, by Euroclone (7 mentions)
L glutamine, by Merck Group (7 mentions)
Sodium pyruvate, by Euroclone (6 mentions)
L glutamine, by Thermo Fisher Scientific (6 mentions)
Albumax, by Thermo Fisher Scientific (6 mentions)
Penicillin streptomycin, by Lonza (5 mentions)
Sodium pyruvate, by Thermo Fisher Scientific (5 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (4 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (4 mentions)
193 protocols using rpmi 1640
1
Co-culture Cytotoxicity Assay for PTEN-Deficient CRC
2
Cell Culture Conditions for Cancer Cell Lines
3
Isolation and Stimulation of Mouse Peritoneal Macrophages
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Resident mouse peritoneal macrophages were isolated from the peritoneal cavity by washing with ice-cold PBS as previously described (21 (link)). Briefly, after centrifugation, the cells were resuspended in RPMI 1640 (Euroclone) supplemented with 5% heat-inactivated fetal calf serum, 50 IU of penicillin/ml, and 50 µg/ml of streptomycin. Cells were then seeded into the wells of 96-well dishes at a density of 5 × 105/well and incubated at 37°C in a 5% humidified CO2 environment. After 24 h, nonadherent cells were removed by washing with medium. Adherent cells were stimulated with increasing multiplicities of infection (MOIs; 1, 5, and 10 µg/ml) of GBS. All infections were carried out by centrifuging bacteria onto cell monolayers for 10 min at 400 × g in order to facilitate bacterial adherence. The number of viable bacteria used in each experiment was carefully determined by plate counting. After incubation at 37°C in 5% CO2 for 25 min, the monolayers were incubated for 18 h in the presence of penicillin (250 IU/ml) and streptomycin (250 µg/ml) to limit the growth of residual extracellular bacteria. In further experiments, cells were stimulated with increasing doses of heat-killed GBS (1, 5, and 10 µg/ml). Cell culture supernatants were collected at 18 h after stimulation to measure cytokine levels.
4
Culturing PSMA-expressing Prostate Cancer Cells
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The PSMA positive (PSMA+) PC3-PIP and PSMA negative (PSMA−) PC3-FLU were kindly provided by Prof Martin G. Pomper (Johns Hopkins Medical School, Baltimore, MD, USA) [57 (link)]. PC3 and LNCaP cell line were purchased from ATCC. LNCaP and PC3 cell lines were cultured until confluence using Ham’s F12 (Euroclone) for PC3 or RPMI-1640 (Euroclone) medium for LNCaP, PC3-PIP and PC3-FLU supplemented with glutamine (2 mM), 10% foetal bovine serum (FBS, Sigma-Aldrich, St. Louis, MO, USA) and penicillin/streptomycin antibiotics (10,000 IU/mL penicillin, 10,000 IU/mL streptomycin, Corning Cellgro, Manassas, VA, USA). PC3-PIP and PC3-FLU cell lines were grown under 20 μg/mL of puromycin to maintain PSMA expression.
5
In Vitro Antimalarial Assay
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The Pf strains D10 (CQ-sensitive), and W-2 (CQ-resistant) were cultured in vitro as described by Trager and Jensen with minor modifications [60 (link)]. Parasites were maintained at 5% hematocrit (human type A-positive red blood cells) in RPMI 1640 (EuroClone, Celbio) medium with the addition of 10% heat inactivate human serum, 20 mM Hepes and 2 mM glutamine (Euroclone). The cultures were maintained at 37 °C in a standard gas mixture consisting of 1–3% O2, 5% CO2, and 92–94% N2. Compounds were dissolved in either water or DMSO and then diluted with medium to achieve the required concentrations (final DMSO concentration <1%, which is non-toxic to the parasite). Asynchronous cultures of Pf with parasitaemia of 1–1.5% and 1% final hematocrit were aliquoted into 96-well flat-bottom microplates (COSTAR) with serial dilutions of test compounds and incubated for 72 h at 37 °C. Parasite growth was determined spectrophotometrically (OD650) by measuring the activity of the parasite lactate dehydrogenase (pLDH) according to a modification of the method of Makler in control and drug-treated cultures [61 (link)]. The antimalarial activity is expressed as 50% inhibitory concentrations (IC50); each IC50 value is the mean and standard deviation of at least three separate experiments performed in duplicate [62 (link)].
6
Cell Culture and Maintenance Protocol
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K562, U937, HL-60, KASUMI, NB4, OCI-AML2, MOLM-14, THP, and KG-1 cells (DMSZ, Germany) were cultured in RPMI 1640 (Euroclone, Italy) supplemented with 10% heat-inactivated fetal bovine serum (FBS) (Sigma-Aldrich, Italy), 2 mM l -glutamine (Euroclone), and antibiotics (100 U/mL penicillin, 100 μg/mL streptomycin, and 250 ng/mL amphotericin-B). HEK293FT, U87MG, HeLa, HCT116, and HCT116 p53−/− cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) (Euroclone) supplemented with 10% FBS, 100 U/mL penicillin/streptomycin (Euroclone), and 6 mM (HEK293FT) or 2 mM glutamine (Euroclone). Transgenic HeLa cells overexpressing GFP-BRD9 were cultured in DMEM supplemented with heat-inactivating FBS, 2 mM l -glutamine, 1% penicillin/streptomycin, 50 μg/mL hygromycin (Thermo Fisher Scientific, Italy), and 2 μg/mL blasticidin S (Sigma-Aldrich).
7
Cell Culture and Synchronization Protocols
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HEK 293T cells (Human Embryonic Kidney 293T, ATCC, Manassas, Virginia, USA) were maintained in Dulbecco’s modified Eagle medium (DMEM, 4.5 g/L glucose, Euroclone, Milan, Italy) supplemented with 10% fetal bovine serum (FBS), antibiotics, L-glutamine and non-essential amino acids (NEAA) (EuroClone) and cultured at 37 °C in 5% CO2. A549 and H1299 cells were grown in RPMI1640 (EuroClone) supplemented with 10% FBS, antibiotics, L-glutamine, and cultured at 37 °C in 5% CO2. All cell lines described in Figure 3 B were grown in accordance with ATCC recommendations and tested for mycoplasma contamination. p53−/− MEFs were a kind gift from P.G. Pelicci and maintained as described previously [31 (link)] For circadian experiments, after 2 h of 50% horse serum containing media serum shock, cells were incubated with serum-free medium for the indicated time.
8
Hypoxia-Induced Breast Cancer Modulation
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Recombinant human IL-1β and BAY 11-7082 were purchased from Pierce Endogen (Rockford, IL) and from Tocris Bioscience (Bristol, UK), respectively. MDAMB231 breast cancer cell line and Topotecan were kindly provided by Dr. Raffaella Giavazzi (IRCCS-Istituto di Ricerche Farmacologiche Mario Negri Milano, Italy). MDAMB231 cells were cultured in RPMI 1640 (Euroclone, Devon, UK), supplemented with 10% FBS (Euroclone) and cultured in atmospheric O2 tension (20% ~140 mmHg), 5% CO2, and 90% humidity at 37°C. The experiments under hypoxia were performed using the workstation INVIVO2 400 (Ruskinn, Pencoed, UK) providing a customized and stable humidified (90%) environment at 37°C through electronic control of O2 (hypoxia: 2% ~14 mmHg) and CO2 (5%) [22 (link)]. Such conditions resemble a mild hypoxic state similar to that observed in breast cancer tissues in vivo [23 (link)].
9
Culturing Lymphoma Cell Lines
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The cutaneous T-cell lymphoma cell line HUT78 and the mantle cell lymphoma cell line GRANTA519 were purchased from Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH and were characterized as specified (https://www.dsmz.de/research/human-and-animal-cell-lines.html ). The B-cell lymphoma cell line WSU-NHL was kindly provided by Dr. M. Introna (Ospedale Riuniti, Bergamo, Italia). All cell lines were cultured in RPMI-1640 supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine, and 100 U/mL penicillin and streptomycin (all purchased from Euroclone). Cells in logarithmic growth phase were used for experiments.
10
Neuronal Cell Line Transfection and Culture
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CAD (mouse catecholaminergic neuronal cell line, Cath.a-differentiated) cells were kindly given by Hubert Laude (Institut National de la Recherche Agronomique, Jouy-en-Josas, France) and cultured at 37 °C in Gibco Opti-MEM (Invitrogen), plus 10% fetal bovine serum and 1% penicillin/streptomycin23 (link). SH-SY5Y cells (neuroblasts from human neural tissue) were cultured at 37 °C in RPMI-1640 (Euroclone), plus 10% fetal bovine serum and 1% penicillin/streptomycin. Transient transfections were performed with Lipofectamine 2000 (Invitrogen) in accordance with the manufacturer’s instructions. CAD cells were transfected in T25 flasks with the appropriate constructs: GFP-tagged constructs, GFP-VASP, GFP-Myo10, or H2B-mCherry for 3 h in serum-free medium and incubated O/N with complete medium. The following day, transfected cells were plated on grids19 (link).
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