HEK-293T cells (E1A-transformed human embryonic kidney cells carrying a temperature sensitive T antigen), were obtained from ATCC/LGC Standards (ATCC, CRL-3216), stored in liquid nitrogen and propagated for a limited number of passages to avoid selection of clonal variants. Cells were routinely maintained in Dulbecco's modified Eagle's medium containing 4.5g/l glucose, 2 mM glutamine, 1 mM sodium pyruvate, nonessential amino acids, penicillin-streptomycin and 10% v/v fetal calf serum (EUROBIO, CVFSVF00–01). Cultures were passaged every 4 or 5 d by trypsinization.
Cvfsvf00 01
Manufactured by Eurobio Scientific
Sourced in France, United States
The CVFSVF00-01 is a laboratory equipment designed for cell culture and related applications. It functions as a ventilated flow cabinet, providing a controlled and sterile environment for handling cell cultures and other sensitive biological materials.
Automatically generated - may contain errors
Lab products found in correlation
Rpmi 1640 medium, by Thermo Fisher Scientific (3 mentions)
G7513, by Merck Group (2 mentions)
Dmem d2429, by Merck Group (2 mentions)
S8761, by Merck Group (2 mentions)
G8769, by Merck Group (2 mentions)
Penicillin streptomycin, by Merck Group (2 mentions)
Be12 702f, by Lonza (2 mentions)
Bt474, by American Type Culture Collection (2 mentions)
Hek293t cell, by American Type Culture Collection (1 mentions)
Htb 20, by American Type Culture Collection (1 mentions)
Sk br 3, by Thermo Fisher Scientific (1 mentions)
Mda mb 231, by Thermo Fisher Scientific (1 mentions)
Mcf 7, by Thermo Fisher Scientific (1 mentions)
Mycoalert detection kit, by Lonza (1 mentions)
Crl 11372, by American Type Culture Collection (1 mentions)
Trypsin edta, by Thermo Fisher Scientific (1 mentions)
Mnng hos, by American Type Culture Collection (1 mentions)
Ham s f 12k, by Thermo Fisher Scientific (1 mentions)
L glutamine, by Thermo Fisher Scientific (1 mentions)
Bhk 21, by American Type Culture Collection (1 mentions)
15 protocols using cvfsvf00 01
1
Propagation of HEK-293T cells
2
Immortalized Rat Hypothalamic Neurons
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rHypoE11 (rat) neuronal cell lines purchased from American Type Culture Collection (ATCC) was conditionally immortalized by transfer of a temperature-sensitive simian virus 40 large tumors (SV40 T) antigen to primary hypothalamic neuronal cell cultures obtained from fetal mice on embryonic days E15, E17, and E18, and from E18 fetal rats [28 (link)]. Cells were cultivated in 1× Dulbecco’s modified Eagle’s medium (DMEM, D5796, Sigma-Aldrich, Saint-Quentin Fallavier, France) with 10% fetal bovine serum (CVFSVF0001, lot: S52751-2262, Eurobio, Courtaboeuf, France), 1% penicillin/streptomycin (P4333, Sigma-Aldrich), and maintained at 37 °C with 5% CO2. For homogeneity, the same serum was used throughout all experiments. The cells grew to form a monolayer culture attached to the culture plate and were split when they reached 70–90% confluence, with a plate ratio of 1:5. Because standard DMEM does not contain vitamin B12, methyl donor deficiency was induced by using a poor medium (DMEM D2429, Sigma-Aldrich) lacking vitamin B9 (folic acid), with the addition of 2 mM glutamine (G7513, Sigma-Aldrich), 3.7% sodium bicarbonate (S8761, Sigma-Aldrich), 0.35% glucose (G8769, Sigma-Aldrich), 10% fetal bovine serum and 1% penicillin/streptomycin. Cells were kept in B9-free conditions for 24 or 48 h before subsequent analyses.
3
Devitalized Human Skin Fibroblast Sheets
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Human Skin Fibroblast (HSFs) isolation and culture were performed in accordance with article L. 1243-3 of the code of public health and under the agreement DC-2008-412 with the University Hospital Center of Bordeaux, France [update 10/10/2014] as previously described [13 (link),14 (link)]. Briefly, HSFs were isolated from adult normal human skin and grown in DMEM/F12 media (#31331-028, Gibco) supplemented with Fetal Bovine Serum (20%; #SH30109 HyClone; #S1810-500 Biowest or #CVFSVF00-01 Eurobio; 1:1). For CAM sheets production, HSFs (passage 5 or 6) were seeded (1 × 104 cells/cm2) in 6-well plates or in 225 cm2 flasks to obtain circular (3.5 cm diameter) or rectangular (10 × 18 cm) sheets. They were cultured for 8 weeks with a supplementation of sodium L-ascorbate (500 μM, #A4034-500 G, Sigma-Aldrich). CAM sheets were rinsed (distilled water), frozen at -80 °C, thawed and then rinsed (distilled water). Large sheets of 108 microns in thickness (± 31 μm, n = 24) were either cut into ribbons (5-mm-wide, with a custom-made automated device), into squares (approximatively 2 × 2 cm, with scissors) or into disks (6-mm-diameter, with biopsy punches) and then air-dried (sterile air flow, at least 2 h). We refer to these steps of freezing / thawing / drying by using the term “devitalization”. All samples were stored at 80 °C until needed.
4
Breast Cancer Cell Line Culture
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Human breast ductal carcinoma cell line BT-474 (American Type Culture Collection (ATCC®) HTB-20™, Manassas, VA, USA) and human breast gland adenocarcinoma cell lines SK-BR-3 (ATCC® HTB-30™, Manassas, VA, USA), MCF-7 (AddexBio C0006008, San Diego, CA, USA), and MDA-MB-231 (AddexBio C0006002, San Diego, CA, USA) were cultured as monolayers in vitro. They were fed with media constituted of Hybri-Care (ATCC® 46-X™) + 0.15% NaHCO3 (BT-474), McCoy’s 5A (ATCC® 30-2007™) (SK-BR-3), MEM (Gibco™ 41090028) (MCF-7), and DMEM 4.5 g/L glucose (Gibco™ 31966021) (MDA-MB-231). All media were supplemented with 10% fetal calf serum (CVFSVF00-01, Eurobio, Courtaboeuf, France) and 1% penicillin–streptomycin mix (Gibco™ 15140122). Cells were cultivated at 37 °C under 95% air and 5% CO2 humidified atmosphere. They were routinely passaged using 0.25% trypsin–EDTA mix (Gibco™ 25200056) and regularly checked for mycoplasma contamination with the MycoAlert™ detection kit (Lonza LT07-218, Basel, Switzerland).
5
Osteoblastic Cell Line Viability Assay
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The osteoblastic cell line hFOB 1.19 (ATCC® CRL11372™, ATCC, USA)77 (link) was cultured using DMEM/F12 (Dulbecco’s Modified Eagle Medium α) (Gibco 10565018) conditioned media supplemented with 10% (V/V) foetal bovine serum (FBS) (Eurobio CVFSVF00-01). Cells were cultured at 37 °C in 5% CO2 in a 10 cm diameter petri dish and trypsinized using 0.05% Trypsin–EDTA (Gibco 25300-054). After sterilization with 70% (w/v) ethanol for 30 min and UV irradiation for 1 h, the filaments and printed scaffolds were dried at room temperature and then placed in contact with hFOB cells for 5 days.
Cell viability was analyzed using MTT assay carried out by incubating 100 µL of 0.5 mg/mL of MTT solution on the cells for 3 h. Purple coloured formazan crystals were dissolved using 100 µL of DMSO (BDH Prolab 23486.297) and the absorbance was recorded at 560 nm using Multiskan plat reader (thermos, USA).
Cell viability was analyzed using MTT assay carried out by incubating 100 µL of 0.5 mg/mL of MTT solution on the cells for 3 h. Purple coloured formazan crystals were dissolved using 100 µL of DMSO (BDH Prolab 23486.297) and the absorbance was recorded at 560 nm using Multiskan plat reader (thermos, USA).
6
Cell Line Culture Conditions
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The A549 (CCL-185, ATCC) lung adenocarcinoma cell line, the LnCaP (89110211, ECACC, Saliibury, UK) prostate adenocarcinoma cell line, the MNNG/HOS (CRL-1547, ATCC, LGC Molsheim, France) osteosarcoma cell line and the U251 (09063001, Sigma-Aldrich, Saint Quentin Fallavier, France) glioblastoma cell line were cultured at 37°C, 5% CO2 and in an environment saturated in humidity. The A549 were cultured in Ham’s F12-K (21127–022, Gibco) complemented with 10% FBS and 2 mM L-Glutamine. The LnCaP were cultured in RPMI complemented (L0501-500, Dutscher) with 10% FBS, 2 mM L-Glutamine and 1 mM sodium pyruvate. The MNNG/HOS were cultured in DMEM 4.5 g/L D-glucose and 0.11 g/L sodium pyruvate (L0106-500, Dutscher, Bernolsheim, France) supplemented with 5% fetal bovine serum (FBS) (CVFSVF00-01, Eurobio Scientific, Les Ulis, France) and 2 mM L-Glutamine (25030–024, Gibco, Paris, France). The U251 were cultured in DMEM 4.5 g/L D-glucose and 0.11 g/L sodium pyruvate complemented with 10% FBS and 2 mM L-Glutamine. All cell lines were regularly tested for the absence mycoplasma.
7
Cell Line Maintenance and Validation
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Wild-type and Irf3−/−Irf7−/− MEF cells were obtained from the Lenschow laboratory (Washington University, USA); BHK21, HFF and Vero cells were purchased at ATCC. All cell lines were tested for mycoplasma, and were cultured in complete Dulbecco modified Eagle's minimal essential medium with high glucose and sodium pyruvate (DMEM, Thermo Fisher Scientific #31966047), supplemented with 10 mM HEPES buffer (Thermo Fisher Scientific #15630056), 1× non-essential amino acids (Thermo Fisher Scientific #11140035), penicillin-streptomycin (Thermo Fisher Scientific #15070063) and 10% fetal calf serum (Eurobio #CVFSVF00-01). Cells were maintained at 37°C and 5% CO2and passaged every 2–4 days at 1/10–1/2 dilutions. Cells were maintained for no more than 6 passages.
8
Characterization of PC3 Cell Line for Cancer Research
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Human PCa PC3 (CRL-1435) cell line (American Type Culture Collection (ATCC)) was received in 2016–2018. This cell line was tested and authenticated by DNA fingerprinting by the ATCC. After reception, cells were amplified in order to make a large reserve of cryopreserved cells. Every 3 months, a new cryopreserved bulb was thawed and used for this study. PC3 cells were cultured in RPMI medium (BE12-702F, Lonza, Levallois-Perret, France), supplemented with 5% FBS (CVFSVF0001, Eurobio, Les Ulis, France) and 1% (v/v) penicillin–streptomycin in a 37 °C-humidified incubator, 5% CO2.
LA (L1876), EPA (17266), and PA (P5177) were from Sigma-Aldrich (Saint-Quentin-Fallavier, France). Calcium channel inhibitors GSK7975A (GLXC03243) and Synta66 (GLXC03244) were from GLIXX Laboratories INC (Hopkinton, MA, USA). The SK3 activator, CyPPA, was purchased from Sigma-Aldrich (Saint-Quentin-Fallavier, France). 1-Ohexadecyl-2-O-methyl-sn-glycero-3-lactose (Ohmline) was synthetized as previously described [28 (link)].
LA (L1876), EPA (17266), and PA (P5177) were from Sigma-Aldrich (Saint-Quentin-Fallavier, France). Calcium channel inhibitors GSK7975A (GLXC03243) and Synta66 (GLXC03244) were from GLIXX Laboratories INC (Hopkinton, MA, USA). The SK3 activator, CyPPA, was purchased from Sigma-Aldrich (Saint-Quentin-Fallavier, France). 1-Ohexadecyl-2-O-methyl-sn-glycero-3-lactose (Ohmline) was synthetized as previously described [28 (link)].
9
Cultivation of Sensitive and Resistant Leukemia Cells
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K562 (ATCC®, CCL-243, described here as K562S to indicate sensitivity to doxorubicin), a human chronic myelogenous leukemia cell line, was obtained from American Type Culture Collection (Manassas, VA, USA). A doxorubicin-resistant cell line (K562R, also known as K562/Adr) was kindly provided by the IRSET institute (Research Institute for Environmental and Occupational Health, INSERM, University of Rennes 1, France). The cells were maintained at 37 °C and 5% CO2 in Gibco™ 1640 Roswell Park Memorial Institute (RPMI-1640) medium containing 10% fetal bovine serum (FBS) (Life TechnologiesTM, Thermo Fisher Scientific, Waltham, MA, USA).
Peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll gradient centrifugation from blood buffy coats of healthy donors, provided by the Etablissement Français du Sang (EFS). The research protocol was conducted under French legal guidelines. After separation of monocytes by 1 h adhesion step, non-adherent PBMCs (peripheral blood lymphocytes, PBL) were harvested. PBL were cultured in RPMI 1640 medium (Gibco, Life technologies, Carlsbad, CA, USA) supplemented with 10% decomplemented fetal bovine serum (CVFSVF00–01, Eurobio), penicillin (100 IU/mL), and streptomycin (100 μg/mL) (15140–122, Gibco). All cells were cultured at 37 °C with 5% CO2.
Peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll gradient centrifugation from blood buffy coats of healthy donors, provided by the Etablissement Français du Sang (EFS). The research protocol was conducted under French legal guidelines. After separation of monocytes by 1 h adhesion step, non-adherent PBMCs (peripheral blood lymphocytes, PBL) were harvested. PBL were cultured in RPMI 1640 medium (Gibco, Life technologies, Carlsbad, CA, USA) supplemented with 10% decomplemented fetal bovine serum (CVFSVF00–01, Eurobio), penicillin (100 IU/mL), and streptomycin (100 μg/mL) (15140–122, Gibco). All cells were cultured at 37 °C with 5% CO2.
10
Conditional Immortalization of Hypothalamic Neuronal Cells
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mHypoE46 (mouse) et rHypoE11 (rat) neuronal cell lines purchased from American Type Culture Collection (ATCC) were conditionally immortalized by transfer of a temperature-sensitive simian virus 40 large tumor (SV40 T) antigen to primary hypothalamic neuronal cell cultures obtained from fetal mice on embryonic days E15, E17 and E18, and from E18 fetal rats [43 (link)]. Cells were cultivated in 1× Dulbecco’s modified Eagle’s medium (DMEM, D5796, Sigma-Aldrich, Saint-Quentin Fallavier, France) with 10% fetal bovine serum (CVFSVF0001, lot: S52751-2262, Eurobio, Courtaboeuf, France), 1% penicillin/streptomycin (P4333, Sigma-Aldrich) and maintained at 37 °C with 5% CO2. For homogeneity, the same lot of serum was used throughout all experiments. The cells grew to form a monolayer culture, attached to the culture plate and were split when they reached 70–90% confluence, with a plate ratio of 1:5. Because standard DMEM does not contain vitamin B12, methyl donor deficiency was induced by using a poor medium (DMEM D2429, Sigma-Aldrich) lacking B9 (folic acid), with addition of 2 mM glutamine (G7513, Sigma-Aldrich), 3.7% sodium bicarbonate (S8761, Sigma-Aldrich), 0.35% glucose (G8769, Sigma-Aldrich), 10% fetal bovine serum and 1% penicillin/streptomycin. Cells were kept in B9-free conditions for 24 or 48 h before subsequent analyses.
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